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ABI PRISM® 373
DNA Sequencer
With XL Upgrade
User’s Manual
© Copyright 2001, Applied Biosystems
For Research Use Only. Not for use in diagnostic procedures.
ABI PRISM and the ABI PRISM design, Applied Biosystems, GeneScan, Genotyper, MicroAmp, Primer Express and
Sequence Navigator are registered trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other
countries.
ABI and AmpliCover are trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries.
All other trademarks are the sole property of their respective owners.
Applied Biosystems
Contents
1 Introduction
1-1
How to Use This Manual
How to Get Started Quickly
Description of ABI PRISM 373 DNA Sequencer with XL Upgrade
Software for Data Analysis
Differences between the ABI PRISM 373 DNA Sequencer with
XL Upgrade and the Model 373
Technical Support
2 Preparing Gels
1-20
1-22
2-1
Polyacrylamide Gels
Preparing Acrylamide Denaturing Gels
Preparing the Plates (all sizes except 48 cm)
Preparing the Gel (all sizes except 48 cm)
Preparing the Plates (48 cm only)
Preparing the Gel (48 cm only)
2-3
2-9
2-13
2-18
2-22
2-25
3 Using the Instrument in Sequencing Mode
3-1
Introduction
Before You Set Up the Instrument
Setting Up the Instrument
Setting Up the Software
Checking the Plates
Preparing and Loading Samples
Observing Instrument Status and Data
Cleaning Up After the Run
Analyzing the Data
Archiving the Data
4 Using the Instrument in GeneScan Mode
Introduction
Before You Set Up the Instrument
Setting Up the Instrument
March 2001
1-3
1-8
1-9
1-16
Contents
3-4
3-5
3-6
3-16
3-27
3-39
3-45
3-47
3-50
3-52
4-1
4-4
4-5
4-9
TOC-iii
Applied Biosystems
Setting Up the Software
Checking the Plates
Preparing and Loading Samples
Observing Instrument Status and Data
Cleaning Up After the Run
Analyzing the Data
Evaluation Checklist
Characteristics of Good Data
Characteristics of Poor Data
Troubleshooting Table
Archiving the Data
5 Using the Data Collection Software
Overview of Data Collection Menu Commands
Using the Data Collection Program
Setting Preferences
Creating and Editing a Sample Sheet
Using the Run Window
Viewing the Data
Controlling the ABI PRISM 373 DNA Sequencer with XL Upgrade
Manually
PMT Gain and Calibration File
Saving and Printing Files
About Data Collection Program Files
6 Instrument Hardware
About the ABI PRISM 373 DNA Sequencer With XL Upgrade
The Loading System
The Separation System
The Scanning/Detection System
Translation Interface Processor Status and Control
7 Computer System
About the ABI Prism 373 DNA Sequencer with XL Upgrade
Computer System
Maintaining and Caring for Your Macintosh Computer
TOC-iv
Contents
4-19
4-30
4-41
4-48
4-50
4-53
4-54
4-55
4-56
4-58
4-65
5-1
5-5
5-12
5-14
5-32
5-40
5-49
5-59
5-61
5-65
5-70
6-1
6-3
6-6
6-10
6-11
6-17
7-1
7-3
7-7
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Applied Biosystems
8 Maintenance/Troubleshooting
Maintenance
Troubleshooting
8-3
8-8
A Applied Biosystems Limited Warranty
A-1
B Quick Reference
B-1
C Sequencing Instrument File
C-1
Sequencing Instrument File
Creating a Matrix File
C-3
C-5
D GeneScan Matrices
D-1
Introduction
Creating a Matrix File
D-3
D-6
E Optimizing PCR
E-1
Preparing Reaction Mixtures
Performing Hot Start PCR
Setting Temperature Control Parameters
Avoiding Contamination
Analyzing Post-PCR Products
March 2001
8-1
Contents
E-3
E-8
E-10
E-12
E-14
TOC-v
000 I'M INVISIBLE
Applied Biosystems
1 Introduction
Contents
How to Use This Manual
3
ABI PRISM 373 DNA Sequencer with XL Upgrade Manual Sets
3
Information Provided in This Manual
4
Overview
4
Model-Specific Notation
4
Special Text Usage
5
User Attention Words
5
Safety Information
6
User Bulletins
7
How to Get Started Quickly
8
Registering Your Software Programs
8
Description of ABI PRISM 373 DNA Sequencer with XL Upgrade
9
Sequencing and GeneScan Analysis on the ABI PRISM 373 DNA Sequencer
with XL Upgrade
10
The Sequencing Applications
11
The GeneScan Applications
11
How the ABI PRISM 373 DNA Sequencer with XL Upgrade Works
12
Electrophoresis and Detection
12
Real-Time Status and Data Observation
12
Data Analysis
13
ABI PRISM 373 DNA Sequencer with XL Upgrade Throughput
14
Software for Data Analysis
16
DNA Sequencing Analysis Software
16
GeneScan Analysis Software
17
Further Analysis of Processed Data
17
The Inherit Sequence Analysis System
17
AutoAssembler Sequence Assembly Software
18
Sequence Navigator Sequence Comparison Software
18
Genotyper Genetic Analysis Software
18
Primer Express Software
18
EditView Software
19
March 2001
1 Introduction
1-1
Applied Biosystems
Differences between the ABI PRISM 373 DNA Sequencer with XL Upgrade and the
Model 373
20
Instrument and Accessories
20
Chemistry
21
Software
21
Technical Support
22
Contacting Technical Support
22
To Contact Technical Support by E-Mail
22
Hours for Telephone Technical Support
23
To Contact Technical Support by Telephone or Fax
23
To Reach Technical Support Through the Internet
27
To Obtain Documents on Demand
28
1-2
1 Introduction
March 2001
Applied Biosystems
How to Use This Manual
IMPORTANT
This manual replaces your previous Model 373 User’s Manual. Transfer
all User Bulletins, the Safety Summary, and personal notations to this
manual before discarding the old manual.
ABI PRISM 373 DNA Sequencer with XL Upgrade Manual Sets
The ABI PRISM 373 DNA Sequencer with XL Upgrade manual set varies with the
configuration of the instrument. The following table shows the manuals you
should receive for the different configurations (this manual is listed in boldface
type).
Table 1-1. ABI PRISM 373 DNA Sequencer with XL Upgrade Reference Manuals
March 2001
For Sequencing Analysis
For GeneScan Analysis
Manual Describes:
ABI PRISM 373 DNA
Sequencer with XL
Upgrade User’s Manual
ABI PRISM 373 DNA
Sequencer with XL
Upgrade User’s Manual
Preparing gels
Running samples
Data collection software
Instrument hardware
Macintosh maintenance
ABI PRISM DNA
Sequencing Analysis
Software User’s Manual
ABI PRISM GeneScan®
Analysis Software User’s
Manual
Software that analyzes data
collected from the instrument
DNA Sequencing
Chemistry Guide
672 GeneScan User’s
Manual
Chemistry protocols for
sample preparation
(Protocols that are supplied
with Sequencing chemistry
kits are not included)
Safety information
Troubleshooting information
User Bulletins
1 Introduction
1-3
Applied Biosystems
Information Provided in This Manual
Overview
This manual describes how to prepare gels and run samples. It provides
information about the data collection software and the instrument and computer
hardware. The manual is equipped with an extensive index and tables of contents
to help you find information quickly. Even if you do not normally read manuals,
these features should help you use it as a reference.
To get started quickly, see How to Get Started Quickly on page 1-8 and refer as
recommended to the appropriate ABI PRISM DNA Sequencing Chemistry Guide and
Sections 2 and 3 of this manual. Appendix B, Quick Reference, contains copies of
useful tables outlining the process of pouring gels and performing runs.
To learn about the instrument and the collection software, read Sections 5
through 7. These sections describe the hardware and software in more depth.
The appendixes contain specialized information, such as the instrument
warranty, a consumables parts list, and details regarding matrices and data files.
This manual is written with the assumption that you have previously used a
Macintosh computer and know how to use the mouse and select items from
menus. If you are new to the Macintosh computer, refer to the Macintosh System 7
Reference manual for information about using it.
Model-Specific Notation
This manual applies to three configurations of Model 373 DNA Sequencers:
•
Classic. This configuration includes 370s with Macintosh upgrade and the
373A. Classic models have electrophoresis chambers which support 24-cm
WTR distances.
•
Leon. This configuration has an electrophoresis chamber which supports
6-, 12-, and 24-cm WTR distances.
•
Stretch. This configuration supports 6-, 12-, 24-, 34-, and 48-cm well-to-read
(WTR) distances and is easily identified by its extended door.
Note
1-4
All three instrument configurations must have the five-filter wheel
capability.
1 Introduction
March 2001
Applied Biosystems
Most of the information in this manual applies to all three configurations,
however, some sections of the manual apply only to one or two of these
configurations. These sections are preceded by the name of the configuration
(Classic, Leon, or Stretch) to which they apply.
Special Text Usage
User Attention Words
The text of this manual includes four user attention words to draw your attention
to safety issues or issues relative to proper operation of the instrument. Each
represents a certain level of attention or action, as described here.
March 2001
Note
Calls attention to information.
IMPORTANT
Indicates information that is necessary for proper instrument operation
or for effective chemistry.
Caution
Indicates that damage to the instrument could result if you do not
comply with this information.
WARNING
Indicates that physical injury to the user or other persons could
result if these precautions are not implemented.
1 Introduction
1-5
Applied Biosystems
Safety Information
Refer to your original Safety Summary for information about operating the ABI
PRISM 373XL safely. The Safety Summary also contains Material Safety Data Sheets
(MSDSs) for chemicals that are commonly used with the instrument. Always keep
the Safety Summary available for reference.
Safety warnings appear throughout the manual at relevant locations. These
warnings address chemical, high voltage, and laser safety and appear in the format
shown below. Please heed the following general warnings:
1-6
WARNING
ELECTRICAL SHOCK HAZARD. The ABI PRISM 373 DNA
Sequencer with XL Upgrade contains a high voltage power supply.
Although the instrument has been designed with safety features in
the door to disconnect the power supply when the door is open,
please follow procedures as prescribed. As with any
electrophoresis apparatus, be careful during instrument operation
and when handling electrodes and liquids.
WARNING
LASER HAZARD. The ABI PRISM 373 DNA Sequencer with XL
Upgrade contains a laser. Operating this instrument without due
regard to the precautions, or in any manner that is not in
compliance with procedures recommended here, may be unsafe
and can cause physical harm from radiation exposure.
WARNING
CHEMICAL HAZARD. The acrylamide and bis-acrylamide used to
prepare sequencing gels for the ABI PRISM 373 DNA Sequencer
with XL Upgrade are toxic chemicals whose use requires utmost
attention to safety. Be sure to read the Material Safety Data Sheets
(MSDSs)for emergency and first-aid procedures. MSDSs are in the
ABI PRISM 373 DNA Sequencer with XL Upgrade Pre-Installation
Manual, P/N 901173. Acrylamide is a neurotoxin that can be
absorbed through the skin. Wear a laboratory coat, eye protection,
and gloves. The handling and disposal of chemical waste is the
responsibility of the user, and not the Applied Biosystems service
representative.
1 Introduction
March 2001
Applied Biosystems
User Bulletins
User Bulletins are updates to this manual that provide a quick way to ensure that
you have current information and new protocols. They contain technical
information that is essential to ABI PRISM 373 DNA Sequencer with XL Upgrade
operation and related laboratory techniques.
March 2001
1 Introduction
1-7
Applied Biosystems
How to Get Started Quickly
Be sure your ABI PRISM 373 DNA Sequencer with XL Upgrade is completely
installed before you begin preparing for a run. After it is installed:
•
Refer to the ABI PRISM DNA Sequencing Chemistry Guide or the 672 GeneScan
User’s Manual as you prepare your DNA template and choose a method for
performing the sequencing or labeling reactions.
•
Use the instructions in Section 3 for step-by-step operation of the
instrument in sequencing mode. Section 3 is extensively cross-referenced
so you can easily refer to Section 5 for more detailed information on the
ABI PRISM 373 DNA Sequencer with XL Upgrade collection software.
•
Use the instructions in Section 4 for step-by-step operation of the
instrument in GeneScan mode. Section 4 is extensively cross-referenced so
you can easily refer to Section 5 for more detailed information on the
ABI PRISM 373XL collection software.
Registering Your Software Programs
When you send in the registration cards for your software programs you become
eligible to receive upgrade notices. Often you also become eligible for technical
support and to purchase software upgrades at lower prices than it would cost to
purchase the new upgraded programs. These privileges might vary, depending on
the company that provides the software.
To register your software, fill out the registration card included in the program
package and return it to the company, as indicated.
1-8
1 Introduction
March 2001
Applied Biosystems
Description of ABI PRISM 373 DNA Sequencer with
XL Upgrade
The ABI PRISM 373 DNA Sequencer with XL upgrade is a high-throughput,
automated instrument system (see Figure 1-1) that is capable of determining base
sequence, fragment size, or relative quantity of fluorescent dye-labeled nucleotide
fragments. It consists of an electrophoresis instrument, a Power Macintosh
Computer that includes software for data collection and data analysis, a
Translation Interface Processor, and an optional printer. Accessories for standard
runs such as combs and spacers are included.
ABI PRISM 373A

Power Macintosh

Macin
Centrtosh
is 650
Translation
Interface
processor
Figure 1-1. The ABI PRISM 373 DNA Sequencer with XL upgrade
Two software applications are available for analyzing the raw data produced by the
instrument. With the Sequencing Analysis software you can analyze DNA
sequence data. The GeneScan® Analysis software allows you to size and quantitate
March 2001
1 Introduction
1-9
Applied Biosystems
DNA fragments. Either or both of these programs is shipped with the instrument,
depending on your order.
Note
This manual describes only how to collect raw data on the instrument.
For information about Sequencing Analysis or GeneScan Analysis
applications, refer to the Sequencing Analysis Software User’s Manual
or the GeneScan Analysis Software User’s Manual, respectively.
Sequencing and GeneScan Analysis on the ABI PRISM 373 DNA
Sequencer with XL Upgrade
IMPORTANT
Analysis of raw data from the ABI PRISM 373 DNA Sequencer with XL
Upgrade requires version 3 or higher of Sequencing Analysis, or
version 2.1 or higher of GeneScan Analysis. The correct version of the
software should be included in your ABI PRISM 373 DNA Sequencer
with XL Upgrade DNA Sequencer upgrade package.
The ABI PRISM 373 DNA Sequencer with XL Upgrade operates the same way for
both Sequencing and GeneScan Analysis modes: it performs electrophoretic
separation and spectral detection of dye-labeled DNA fragments. The difference
between the two modes is in the preparatory chemistries and in the analysis
performed on the resulting data.
Sequencing and GeneScan Analysis software programs each analyze the resulting
data for your application. The analysis software programs are briefly described on
page 1-16, and more completely described in the separate analysis software user’s
manuals.
To prepare a DNA template, you can use standard laboratory methods. For
example, you can use PCR or clone the DNA into a vector such as M13 or pUC,
amplify the vector, then purify the template by standard methods. To prepare
samples for running on the ABI PRISM 373 DNA Sequencer with XL Upgrade, you
must incorporate fluorescent labels. The chemistries for incorporating
fluorescent labels are different, depending on whether your application is
Sequencing or GeneScan Analysis.
1-10
1 Introduction
March 2001
Applied Biosystems
The Sequencing Applications
For sequencing applications on the ABI PRISM 373 DNA Sequencer with XL
Upgrade, the sequencing reaction involves the incorporation of fluorescent
labels, instead of radioactive isotopes. Four different dyes identify the A, C, G, and
T extension reactions. You can incorporate the dye labels using either 5'-dye
labeled primers or 3'-dye labeled dideoxynucleotide terminators.
You can use standard polymerases, such as AmpliTaq® and Sequenase T7 DNA
polymerase, for primer extension. Both are available in kits with the dye
terminator and the dye primer chemistries from Applied Biosystems. The labeling
method you use depends on the sequencing strategy and on your personal
preference. Refer to the ABI PRISM DNA Sequencing Chemistry Guide.
The GeneScan Applications
GeneScan analysis of Sample files includes establishing a baseline, adjusting for
spectral overlap of the dyes, peak detection, and size calling.
When you use the GeneScan system on the ABI PRISM 373 DNA Sequencer with
XL Upgrade, you can label DNA fragments with up to three different fluorescent
dye colors, and then use a fourth dye color to label an internal lane or size
standard. When you use an internal size standard, the GeneScan software
performs precise size calling without band-shift artifacts and run-to-run variation,
problems often encountered using other techniques. GeneScan Analysis software
can also be used for relative quantitation when a dye-labeled fragment of known
quantity is included in the same lane as the DNA fragments.
You can display the results of an experiment as electropherograms, tabular data,
or a combination of both. Electropherograms are a graphical display of peaks
detected during collection. Each electropherogram represents a single lane for
ABI PRISM 373 DNA Sequencer with XL Upgrade data. The tabular data provides
detailed sizing and quantitative information.
Fluorescent labels are incorporated in the form of 5'-end labeling using
NHS-esters or phosphoramidites. A second labeling strategy incorporates internal
dye labels in the form of fluorescent dNTPs
March 2001
1 Introduction
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Applied Biosystems
How the ABI PRISM 373 DNA Sequencer with XL Upgrade
Works
After your DNA fragments are labeled, the ABI PRISM 373 DNA Sequencer with
XL Upgrade automatically separates and detects them, providing data for analysis
by the Sequencing Analysis or GeneScan Analysis software program.
Electrophoresis and Detection
DNA templates labeled with four different fluorescent dyes are combined and
loaded into one lane on a vertical acrylamide slab gel. You can analyze up to 66
lanes simultaneously on one gel.
The dye-labeled DNA fragments electrophorese through the acrylamide gel and
separate according to size. At the lower portion of the gel they pass through a
region where a laser beam continuously scans across the gel. The laser excites the
fluorescent dyes attached to the fragments, and they emit light at a specific
wavelength for each dye. The data collection software collects the light intensities
using the 5-filter wheel and stores them as electrical signals for eventual
processing.
Real-Time Status and Data Observation
The data collection program stores the digitized output signal into files on the
Macintosh hard disk. The computer screen displays the information received
from the ABI PRISM 373 DNA Sequencer with XL Upgrade in real time.
Note
Real-time viewing of the first peaks as they pass the laser detection site
occurs within 90 minutes of loading the samples on the gel during a run
using 24- or 34-cm separation distance.
You can view instrument status and data in several windows on the computer
screen by simply choosing the window name from the Window menu.
1-12
•
The Status window displays current instrument conditions and the set
values for the electrophoresis power supply.
•
The Electrophoresis History window displays the actual values for the
electrophoresis power supply and gel temperature.
1 Introduction
March 2001
Applied Biosystems
•
The Scan window displays the raw data; it shows the sweeps of the laser
across the gel with a different colored line representing light emission
through each filter.
•
The Gel window displays a reconstruction of actual data as it would appear
on an autoradiogram.
Data Analysis
At the end of collection, the computer can automatically analyze the collected
data and print electropherograms on a color printer. Depending on your
application, this is performed by either the Sequencing Analysis or the GeneScan
Analysis software. Both programs are briefly described on page 1-10, and more
completely described in the separate analysis software user’s manuals.
You can display the raw and analyzed data on the computer screen, or edit and
print it at any time.
March 2001
1 Introduction
1-13
Applied Biosystems
ABI PRISM 373 DNA Sequencer with XL Upgrade Throughput
You can use the ABI PRISM 373 DNA Sequencer with XL Upgrade in different ways
for various combinations of accuracy, read length, and number of channels.
Table 1-2 summarizes the throughput for typical analysis runs.
Table 1-2. ABI PRISM 373 DNA Sequencer with XL Upgrade Throughput for
Sequencing
Instrument WTR
Gel
Model
Length Concen(cm)
tration
(%)
Scan Mode
Gel
Thickness
(mm)
Classic
24
6.0
0.4
Classic
24
4.75
Leon
24
6.0
Leon
24
Stretch
24
Stretch
Stretch
Power Collec- Length of
(W) tion Time
Read
(hr)
(bases
resolved)
XL or Full scan
30
12–14
450
0.4
BaseSprinter
0.4
XL or Full Scan
40
6.5
450
30
12–14
450
4.75
0.4
BaseSprinter
40
6.0
0.4
XL or Full Scan
24
4.75
0.4
BaseSprinter
34
4.75
0.4
XL or Full Scan
Stretch
34
4.25
0.4
BaseSprinter
Stretch
48
4.0
0.3
XL or Full Scan
261
35
321
45
401
6.5
450
12–14
450
7
450
14–16
550
8
550
17–18
700
1. If necessary, adjust the power to achieve spacing of 9.5 to 12 for analyzed data.
1-14
1 Introduction
March 2001
Applied Biosystems
Table 1-3. ABI PRISM 373 DNA Sequencer with XL Upgrade Throughput for
GeneScan
March 2001
Application
Resolution Size Standard Gel Type
Gel
Run
(bp)
(Acrylamide) Length Time
(cm)
(hr)
Microsatellite Genotyping
(Linkage Mapping Set,
dinucleotide tepeats)
1
GS-350 or
GS-500
6.0%
denaturing
24
6
Microsatellite Genotyping
(tri- and tetranucleotides)
1–2
GS-500
6.0%
denaturing
12
4
VNTRs
1–2
GS-350 or
GS-500
8.0%
denaturing
12
4
Large Fragments
(0.5–2.5 kb)
3–4
GS-1000 or
GS-2500
8.0%
denaturing
6
2
AFLP™ Plant Mapping Kit
1
GS-500
6.0%
denaturing
24
11
Stock Marks™ for Cattle
1
GS-350
6.0%
denaturing
24
6
AmpFlSTR™
(tetranucleotides)
1
GS-500
6.5%
denaturing
24
6
1 Introduction
1-15
Applied Biosystems
Software for Data Analysis
Two standard software programs are available to analyze raw data from the
ABI PRISM 373XL: Sequencing Analysis and GeneScan Analysis.
DNA Sequencing Analysis Software
The Sequencing Analysis software analyzes raw sequencing data collected by the
data collection program on the ABI PRISM genetic analysis instruments.
The analysis software processes data using a multicomponent analysis, baseline
subtraction and scaling. After processing, it detects the peaks and determines the
sequence. The four-dye, one-lane sequencing approach eliminates
electrophoresis variables and reduces lane-to-lane variations.
You can reanalyze and edit the sequence data in a format called a Sample file. The
same Sample file can be used by other Applied Biosystems software programs for
additional data processing. Also, Sample files are in formats that you can export
to commercially available or user-generated programs on the Macintosh and on
other compatible computers for further analysis.
The Sequencing Analysis program comes bundled with three other programs,
GelDocII, DataUtility, and Factura. GelDocII removes tracking data from a Gel file
and removes and restores the gel image from a Gel file. The DataUtility program
performs two functions. It makes matrices for use with the ABI PRISM 373 DNA
Sequencer with XL Upgrade Collection and Sequencing Analysis software, and
monitors noise levels for troubleshooting by Applied Biosystems technical
specialists. Factura performs additional processing on Sample files to clarify the
target DNA sequence.
1-16
1 Introduction
March 2001
Applied Biosystems
GeneScan Analysis Software
The GeneScan® Analysis software enables you to use the automated fluorescence
detection capabilities of the ABI PRISM genetic analysis instruments to determine
accurate sizing and relative quantitation of DNA fragments. You can size PCR
products (STRs, VNTRs, and RAPDs) and perform quantitative gene analysis
(COP assays, mRNA) faster than with traditional methods, yet with sufficient
resolution to accurately analyze molecular fragment lengths in the 50–500
nucleotide size range. The software uses internal lane size standards to reduce
gel-to-gel and lane-to-lane variation.
The software automates the entire process of separating, quantifying, and sizing
DNA fragments. The data can be displayed as a gel image, an electropherogram,
tabular data, or a combination of electropherograms and the corresponding
tabular data.
Further Analysis of Processed Data
The Inherit Sequence Analysis System
Inherit is a multi-user system used to edit, assemble, and analyze DNA and protein
sequence data. The Inherit programs are designed to run on the Macintosh
platform either independently or interactively with a UNIX server. They provide
a robust set of tools for sequence analysis and allow you to access and store data in
a common file format.
Inherit has three Macintosh programs:
March 2001
•
Factura prepares sequence data for analysis and alignment. It identifies
specified vector and ambiguity ranges, restriction sites, and a specified
confidence range. It also identifies multiple base positions with codes
described by the International Union of Biochemists (IUB codes), based
on a user-defined threshold. The program processes sequences in batches,
speeding the process considerably.
•
AutoAssembler™ allows you to quickly and efficiently assemble small
overlapping pieces of DNA into larger segments of DNA, using ABI PRISM
373 DNA Sequencer with XL Upgrade data as well as other types of data. It
provides powerful tools for editing the sequences, including the ability to
display constantly-spaced electropherograms that are synchronized with
the assembled sequences. You can build a consensus from the assembled
sequences and export it for use with other programs.
1 Introduction
1-17
Applied Biosystems
•
GeneAssist allows you to analyze data from ABI PRISM 373 DNA Sequencer
with XL Upgrade sequence files, files processed in AutoAssembler, text
files, or a database. You can use this program to rapidly search biological
databases for sequence similarity or patterns, and to interactively work with
the resulting sequence lists. In addition to searching results, the program
can create dot plots, alignments, and restriction maps of sequence data.
AutoAssembler Sequence Assembly Software
AutoAssembler is available as a separate program from the Inherit system. You can
purchase it for use with a Macintosh only, and perform the same functions as
described above, using a local algorithm. Upgrade to the more powerful assembly
capabilities of the Inherit system by adding the client-server option (which runs
on a UNIX server). This provides a multi-user environment and allows you to use
an assembly algorithm that handles repeat sequences and large projects more
efficiently than the local algorithm.
Sequence Navigator Sequence Comparison Software
Sequence Navigator™ software is specifically designed to address the needs of
researchers performing comparative sequencing, as it accurately aligns sequences
to a standard or a population and identifies sequence variants.
Sequence Navigator runs on the Macintosh and can be used for many
applications, such as mutation identification/heterozygote screening of
sequences such as p53 and HIV, and mitochondrial DNA. The program
incorporates five powerful algorithms for pairwise or multiple alignment of DNA
and protein sequences. It is bundled with the Factura program described above,
allowing identification of heterozygous base positions and quick cleanup of
sequences before alignment.
Genotyper Genetic Analysis Software
Genotyper™ is a data analysis and transformation tool that converts data from
GeneScan into user application results. Genotyper converts data into the format
required by downstream applications, such as linkage analysis programs,
databases, or spreadsheets. The Genotyper software thereby helps create seamless
automation between the ABI PRISM 373 DNA Sequencer with XL Upgrade and the
final experimental results.
Primer Express Software
Primer Express™ is a primer design program with an easy-to-use interface. The
software is applications oriented, taking into consideration the most updated
criteria for primer design.
1-18
1 Introduction
March 2001
Applied Biosystems
EditView Software
EditView is a free, DNA Sequence viewer that allows you to view and print
sequence data from an ABI PRISM 373 DNA Sequencer with XL Upgrade, an ABI
PRISM 377 DNA Sequencer, or an ABI PRISM 310 Genetic Analyzer. Using EditView
on your Macintosh computer, you can open an analyzed sample file and view the
sequence data either as an electropherogram (traces), or in text format. You can
then edit individual bases, export the data to a text file, or print. To receive a copy
of EditView, visit our Web site at www.appliedbiosystems.com/techsupport.
March 2001
1 Introduction
1-19
Applied Biosystems
Differences between the ABI PRISM 373 DNA
Sequencer with XL Upgrade and the Model 373
Instrument and Accessories
• More samples per instrument run. The Model 373 allows 36 samples to be
analyzed simultaneously. With the ABI PRISM 373 DNA Sequencer with XL
Upgrade, you can analyze up to 64 samples at one time. This increased rate
of throughput is due to the redesign of the rate at which the laser detects
data.
• Instrument Performance. While more samples can be analyzed
simultaneously, parameters such as length of sequence detected, peak
sizing and quantitation, and time allocated to run the instrument is not
expected to change after the ABI PRISM 373 DNA Sequencer with XL
Upgrade upgrade is installed.
•
New spacers and combs. The XL combs (48- and 64-well sharks-tooth and 50and 66-well square-tooth) are made of a material called Mylar. In addition
to the combs, glass plate spacers, casting combs, and overlays are also
available in Mylar. The other combs supplied by Applied Biosystems for use
on the ABI PRISM 373 DNA Sequencer with XL Upgrade are made of a
material called Valox.
IMPORTANT
•
Always use combs, glass plate spacers, and casting combs as a
matched set. Do not mix and match material types or thicknesses within
a set. Mismatching can cause loose-fitting combs, and can result in
sample leaking and/or damage to the gel.
New glass plate spacer design. New glass plate spacers are made of Mylar to be
compatible with the XL combs. The new spacers are available in only one
length but are marked to indicate where to cut them in order to fit each
size of glass plate. Offering spacers in a single length reduces the chances
of making errors during order entry. It also allows us to offer them at a
lower price than previous custom-cut spacers.
Notches are cut out of one end of the spacers. When you assemble the
plates, be sure that the notches are at the top of the plates and facing
1-20
1 Introduction
March 2001
Applied Biosystems
toward the well. These new spacers help reduce buffer leaks between the
gel and spacer.
•
Power Macintosh control of instrument. The instrument is operated entirely
from the Power Macintosh computer. While the Model 373 instrument was
controlled by a combination of the keypad on the front of the instrument
and the computer, the keypad on the front of the instrument is now used
only for instrument diagnostics.
•
New user’s manual. The new manual is a smaller, more convenient size, and
contains a glossary, a quick reference procedure, and an expanded index.
•
Translation Interface Processor. This hardware acts as a translator and memory
buffer between the ABI PRISM 373 DNA Sequencer with XL Upgrade and
the Power Macintosh. This enables the instrument to be controlled by the
Power Macintosh. In addition, the Translation Interface Processor
increases the amount of memory buffer available with the Model 373.
Chemistry
•
Load less sample. Smaller well size requires less sample. See Table 3-3 on
page 3-40 for Sequencing sample volumes. See Table 4-2 on page 4-43 for
GeneScan application load volumes.
Software
March 2001
•
Streamlined data collection software. Input of information and viewing of
real-time data/instrument status is simplified. All data for a run can be
viewed in the Gel window during data collection.
•
Compatibility with Analysis software. Raw data generated by the ABI PRISM 373
DNA Sequencer with XL Upgrade can be analyzed using only version 3 or
higher of Sequencing Analysis software or version 2.1 or higher of
GeneScan Analysis software.
1 Introduction
1-21
Applied Biosystems
Technical Support
Contacting Technical Support
You can contact Applied Biosystems for technical support by telephone or fax, by
e-mail, or through the Internet. You can order Applied Biosystems user
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To Contact Technical Support by E-Mail
Contact technical support by e-mail for help in the following product areas:
1-22
Product Area
E-mail address
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[email protected]
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Peptide and DNA Synthesis
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or
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1 Introduction
March 2001
Applied Biosystems
Hours for Telephone Technical Support
In the United States and Canada, technical support is available at the following
times:
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Hours
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Dial...
Fax
Dial...
ABI PRISM® 3700 DNA
Analyzer
1-800-831-6844,
then press 8
1-650-638-5981
1-800-831-6844,
1-650-638-5981
DNA Synthesis
then press 21
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1-800-831-6844,
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then press 22
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(includes GeneScan®
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then press 23
Integrated Thermal Cyclers
(ABI PRISM ® 877 and Catalyst
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then press 24
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then press 26
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SQL GT™ applications)
March 2001
1-800-831-6844,
1-800-831-6844,
1-505-982-7690
then press 25
1 Introduction
1-23
Applied Biosystems
Product or
Product Area
Telephone
Dial...
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then press 31
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6 for the 6700
or dial 1-800-831-6844,
then press 5
1-240-453-4613
Voyager MALDI-TOF
Biospectrometry and Mariner
ESI-TOF Mass Spectrometry
Workstations
1-800-899-5858,
then press 13
1-508-383-7855
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Workstations and Poros
Perfusion Chromatography
Products)
1-800-899-5858,
then press 14
1-508-383-7855
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Synthesis Systems
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then press 15
1-508-383-7855
Peptide Synthesis (Pioneer
and 9050 Plus Peptide
Synthesizers)
1-800-899-5858,
then press 15
1-508-383-7855
PNA Custom and Synthesis
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then press 15
1-508-383-7855
FMAT 8100 HTS System and
Cytofluor 4000 Fluorescence
Plate Reader
1-800-899-5858,
then press 16
1-508-383-7855
Chemiluminescence (Tropix)
1-800-542-2369 (U.S.
1-781-275-8581
only),
or 1-781-271-0045
Applied Biosystems/MDS Sciex
1-24
1-800-952-4716
1 Introduction
1-650-638-6223
March 2001
Applied Biosystems
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March 2001
1 Introduction
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Applied Biosystems
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Dial...
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Dial...
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Tokyo)
81 3 5566 6230
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1-26
305-670-4350
1 Introduction
305-670-4349
March 2001
Applied Biosystems
To Reach Technical Support Through the Internet
We strongly encourage you to visit our Web site for answers to frequently asked
questions and for more information about our products. You can also order
technical documents or an index of available documents and have them faxed or
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To submit technical questions from North America or Europe:
Step
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experts within 24 to 48 hours.
March 2001
1 Introduction
1-27
Applied Biosystems
To Obtain Documents on Demand
Free, 24-hour access to Applied Biosystems technical documents, including
MSDSs, is available by fax or e-mail or by download from our Web site.
To order
documents...
Then...
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1-28
1 Introduction
March 2001
100 I'M INVISIBLE
Applied Biosystems
2 Preparing Gels
Contents
Polyacrylamide Gels
Mechanism of Polymerization
Effect of Reagent Purity on Gel Quality
Acrylamide
Bis-acrylamide
Ammonium Persulfate (APS)
TEMED
Urea
Buffers and Gel Additives
Other Factors that Affect Gel Quality
Rate of Polymerization
Air bubbles
Using an Old Gel
Preparing Acrylamide Denaturing Gels
Reagents and Supplies for Stock Solutions
10X TBE Stock Solution
40% (19:1) Acrylamide Stock Solution
Reagents and Supplies for Gels
Preparing the Plates (all sizes except 48 cm)
Cleaning the Plates
Applying the Bind-Silane Compound
Assembling the Plates
Preparing the Gel (all sizes except 48 cm)
Preparing Acrylamide-Urea Gel Solutions
Preparing 4.25%, 4.75%, or 6% Acrylamide-urea Gel Solutions
Casting the Gel (all sizes except 48 cm)
Preparing the Plates (48 cm only)
Preparing Gel Casting Equipment for a 48 cm Run
Preparing Glass Plates for 48 cm Gels
Applying the Bind-Silane Compound
Assembling the Glass Plates for a 48 cm Run
March 2001
2 Preparing Gels
3
3
3
3
4
5
5
6
6
6
6
7
8
9
10
10
11
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13
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15
18
18
18
20
22
22
22
23
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2-1
Applied Biosystems
Preparing the Gel (48 cm only)
Preparing 4% Acrylamide-urea Gel Solutions
Casting the 48-cm Gel
2-2
2 Preparing Gels
25
25
26
March 2001
Applied Biosystems
Polyacrylamide Gels
For sequencing, the polyacrylamide gel is one of the most important variables that
determines the number of bases you can read. For fragment analysis, the
polyacrylamide gel is critical for the resolving capabilities. In short, your data is
only as good as the quality of your gel.
The quality of your gel is even more important with the ABI PRISM 373XL than it
is with manual sequencing, since much more DNA (over 600 bands) passes
through a gel in a single loading on the ABI PRISM 373XL. Therefore, it is of
paramount importance to produce a first-rate gel. Some of the factors that affect
gel quality are described below.
Mechanism of Polymerization
Polyacrylamide gels are formed by the copolymerization of acrylamide and
bis-acrylamide. The process begins when TEMED (tetramethylethylenediamine)
reacts with APS (ammonium persulfate) to yield a TEMED radical cation, a sulfate
radical and a sulfate ion. The sulfate radical adds an unpaired electron to the
acrylamide monomer, converting it to a free radical which then reacts with more
monomer, causing polymer chain elongation. The polymer chains are randomly
cross-linked with bis-acrylamide to form the gel matrix.
Effect of Reagent Purity on Gel Quality
It is extremely important that you use pure gel reagents from a reliable source.
Optimal results depend on gel quality and therefore, on gel reagent quality.
Acrylamide
Impurities in this reagent (or in bis-acrylamide) can cause:
•
Irreproducible gel porosity
•
Deviant mobility
•
Inhibition of polymerization
•
Poor resolution
all of which lead to poor results, wasted time, and frustration.
March 2001
2 Preparing Gels
2-3
Applied Biosystems
These problems lead to compromised reproducibility. Some possible
contaminants that can cause them include the following:
•
Acrylic acid - The hydrolysis product of acrylamide. It copolymerizes with
acrylamide and bis-acrylamide. Its presence causes the DNA to migrate
slowly, resulting in broad, diffuse bands and consequently, poor resolution.
•
Linear polyacrylamide - Caused by catalytic contaminants in the dry
acrylamide monomer. It decreases the effective concentration of the
acrylamide, causing the DNA to migrate faster.
•
Ionic contaminants - Mostly metals, such as iron or copper. These can
inhibit or accelerate polymerization.
Good quality solid form/dry acrylamide can be stored at room temperature for up
to one year. You can store a 40% stock solution of acrylamide and bis-acrylamide
(19:1) at 4 °C for up to one month.
WARNING
CHEMICAL HAZARD. Acrylamide and bis-acrylamide are
neurotoxins. Avoid inhalation and skin contact. Wear gloves at all
times and work in a fume hood when handling acrylamide
solutions, and use appropriate precautions to avoid inhalation of
crystalline acrylamide. Read the MSDS in the Safety Summary
provided with your original user’s manual.
Bis-acrylamide
Impure bis-acrylamide contains some of the same contaminants as acrylamide.
Store it dry at room temperature for up to one year. You can store a 40% stock
solution of acrylamide and bis-acrylamide (19:1) at 4 °C for up to one month.
Note
2-4
You can either make 40% acrylamide stock solution from solid
acrylamide and bis-acrylamide, or buy high-quality premixed,
preweighed powders and dilute them to a 40% solution. Use distilled or
deionized water only, and store at 4 °C for up to one month. Using the
pre-made powders saves time and minimizes exposure to the solid
form of the neurotoxins acrylamide and bis-acrylamide.
2 Preparing Gels
March 2001
Applied Biosystems
Ammonium Persulfate (APS)
APS (ammonium persulfate) begins to decompose at once when dissolved in
water. The result is loss of activity. For reproducible results, therefore, you should
prepare APS solutions fresh daily. It is vital that this chemical be fresh since it
affects the rate of polymerization and thus the gel properties. Store solid APS
tightly sealed, since it is hygroscopic. If tightly sealed, it can be stored at 4 ˚C for
up to 1 year.
Note
Store solid APS in an airtight container with desiccant, to keep it dry. To
avoid water condensation problems, let the bottle of APS warm to room
temperature before opening it.
TEMED
TEMED (N, N, N', N'-tetramethylethylenediamine) is by nature very reactive and
prone to oxidation. The oxidized form is yellow and less active. Using oxidized
TEMED slows gel polymerization time, thereby significantly altering the gel
characteristics. Because it is hygroscopic, this initiator gradually accumulates
water, which increases the rate of oxidation. Water-free TEMED (>99% pure),
stored in a tightly sealed container at room temperature, should be good for up
to six months.
WARNING
March 2001
CHEMICAL AND FIRE HAZARD. TEMED is extremely flammable
and can be very destructive to the skin, eyes, nose, and
respiratory system. Keep it in a tightly closed container. Avoid
inhalation and contact with skin, eyes and clothing. Always work
under a hood and wear chemical resistant gloves when handling
TEMED solutions. Read the MSDS in the Safety Summary
provided with your original user’s manual.
2 Preparing Gels
2-5
Applied Biosystems
Urea
Use urea in the crystalline form, and weigh it fresh each time you use it. Old urea
has breakdown products that affect sample migration.
WARNING
CHEMICAL HAZARD. Urea is a potential mutagen. Avoid
inhalation and contact with skin, eyes and clothing. Read the
MSDS in the Safety Summary provided with your original user’s
manual.
Note
Do not make up a stock solution containing the urea. Prepare the
components fresh.
Buffers and Gel Additives
Metals, non-buffer ions, and decomposition products are contaminants
commonly found in reagents such as Tris, borate and urea. Transition metal ions
tend to affect the polymerization in various ways, causing run-to-run
irreproducibility. Non-buffer ions tend to inhibit DNA mobility and also cause
run-to-run irreproducibility. To help avoid these problems, work only with high
quality Tris, borate and urea, and use only distilled or deionized water for diluting
reagents.
Other Factors that Affect Gel Quality
Rate of Polymerization
The properties of the gel depend on the rate of polymerization. The rate of
polymerization is affected by temperature, initiator concentration, and oxygen.
Each parameter is discussed in detail in this section for academic reasons only.
The protocol in this section has already been optimized with regard to
temperature, initiator concentration and oxygen content, therefore, you do not
need to experiment with these variables.
Temperature: Controlling the temperature is crucial for achieving reproducible
gels because it directly affects the polymerization time and thus affects the gel
properties. A gel formed in a cold environment (at 4 °C) is turbid, porous, and
inelastic, and the run-to-run reproducibility is greatly compromised.
Polymerization in a 20–23 °C room (“normal” room temperature) is optimal. It is
important that the gel solution and glass plates as well as the room temperature
2-6
2 Preparing Gels
March 2001
Applied Biosystems
be at 20-23 °C. Gels formed at this temperature are transparent, less porous, more
elastic, and more reproducible. If the temperature is too high, the polymer chains
are shorter and the resulting gel is inelastic, again resulting in non-reproducibility
from run to run.
Initiator Concentration: Increasing the concentration of TEMED or APS
decreases the average polymer chain length, and increases gel turbidity.
Decreasing the concentration of TEMED or APS increases the average polymer
chain length, and decreases gel turbidity.
Decreasing the concentration of initiators has a desirable effect, except that this
also causes slower polymerization. When polymerization is too slow, oxygen can
enter the monomer solution during the process and inhibit polymerization.
If you use degraded TEMED or APS, the concentration of initiators is less than
that recommended in the protocol.
Oxygen: Oxygen acts as a free radical trap, thereby inhibiting polymerization. The
result is a porous gel. To prevent the problems caused by oxygen, be careful to
meet the following conditions.
•
Polymerization must be fast enough to prevent too much oxygen from
dissolving into the gel solution during polymerization. As long as you use
fresh, high-quality reagents and follow the protocol in this manual, oxygen
should not cause a problem during polymerization.
•
Minimize the amount of oxygen dissolved in the gel solution prior to
casting the gel, since it can interfere with the rate of polymerization. Partial
degassing is accomplished during the vacuum filtration step of this
protocol. It is important to keep the vacuum strength and time constant
during this step for run-to-run reproducibility. Since cold solutions have a
greater capacity for dissolved oxygen, perform vacuum filtering (and gel
casting) with the solution at room temperature. Also, be gentle during the
stirring and pouring steps of gel casting so that oxygen is not introduced.
Air bubbles
Air bubbles can distort the sample path, which affects lane tracking. Pour gels
carefully and gently so bubbles do not form. If bubbles are trapped between the
comb and the gel solution, carefully remove and reinsert the comb until no
bubbles are trapped.
March 2001
2 Preparing Gels
2-7
Applied Biosystems
Using an Old Gel
For consistent results, use gels between two and six hours after casting. Be sure to wait at
least two hours after casting the gel to ensure complete polymerization, but do not
wait longer than six hours. After six hours resolution begins to noticeably
deteriorate. Gels that stand overnight can show significantly slower DNA
migration, due to the slow hydrolysis of urea to ammonium carbonate.
Since the amide groups of the polymer slowly hydrolyze into acid groups, gels that
stand 48 hours can also show significant loss in resolution beyond 350 bases.
WARNING
2-8
CHEMICAL HAZARD. The acrylamide and bis-acrylamide used to
prepare sequencing gels for the ABI PRISM 373 DNA Sequencer
with XL Upgrade are toxic chemicals whose use requires utmost
attention to safety. Be sure to read the Material Safety Data Sheets
(MSDSs)for emergency and first-aid procedures. MSDSs are in the
ABI Prism 373 DNA Sequencer with XL Upgrade Pre-Installation
Manual, P/N 901173. Acrylamide is a neurotoxin that can be
absorbed through the skin. Wear a laboratory coat, eye protection,
and gloves. The handling and disposal of chemical waste is the
responsibility of the user, and not the Applied Biosystems service
representative.
2 Preparing Gels
March 2001
Applied Biosystems
Preparing Acrylamide Denaturing Gels
This protocol for the preparation of acrylamide gels follows generally accepted
procedures; however, there are some important distinctions to remember
throughout the process. The ABI PRISM 373XL DNA Sequencer is a real-time
electrophoresis detector. A laser scans across a region of the gel during the
electrophoretic process, detecting the fluorescently labeled DNA passing through
that region. The glass and the gel must be non-fluorescent. Be especially careful
to:
•
Eliminate any particulate or fluorescent matter from the acrylamide gel
solutions.
•
Keep the glass plates extremely clean and free of dust.
•
Always use ultra-pure reagents and high-grade, distilled, deionized water to
prepare solutions
•
Wear clean laboratory gloves.
•
Filter all solutions to remove any particulate matter that may fluoresce or
scatter light.
To maximize gel resolution, high quality acrylamide gels are essential, particularly
when you are sequencing templates of greater than 350 bases in length. If you
prepare acrylamide solutions with low-quality starting materials or heat them
excessively, breakdown products might detract from the quality of the run. Using
a deionizing resin and preparing carefully helps to avoid problems caused by such
breakdown activity.
WARNING
March 2001
CHEMICAL HAZARD. Acrylamide and bis-acrylamide are
neurotoxins. Avoid inhalation and skin contact. Wear gloves at all
times and work in a fume hood when handling acrylamide
solutions, and use appropriate precautions to avoid inhalation of
crystalline acrylamide.
2 Preparing Gels
2-9
Applied Biosystems
Reagents and Supplies for Stock Solutions
You can either make 40% acrylamide stock solution from solid acrylamide and
bis-acrylamide, or buy high-quality premixed, preweighed powders and dilute
them to a 40% solution. To dilute them use distilled or deionized water only.
Using the pre-made powders saves time and minimizes exposure to the solid form
of the neurotoxins acrylamide and bis-acrylamide. You can also purchase
ready-made stock solutions of the 10X TBE buffer. The concentration must be
0.89 M Tris-base, 0.89 M Borate, 0.02 M EDTA, pH 8.3 ± 0.2.
If you prefer to prepare your own stock solutions, the recipes follow. Be careful to
use ultra-pure reagents and high-grade, distilled, deionized water to prepare the
solutions.
10X TBE Stock Solution
Per liter:
•
Tris base
108.0 g
•
Boric Acid
•
Na2EDTA • 2H2O
55.0 g
8.3 g
Working solution (1X) is 89 mM Tris-base, 89 mM Boric acid, 2 mM EDTA;
pH≈ 8.3 at ambient temperatures.
IMPORTANT
Discard the working solution if the pH is not 8.3 (± 0.2), and make a
fresh solution. Do not attempt to adjust the pH.
To prepare the 1X TBE working solution (Classic and Leon):
1.
Add 150 mL 10X TBE stock solution to a large graduated cylinder.
2.
Dilute with dH2O to a total volume of 1500 mL.
To prepare the 1X TBE working solution (Stretch):
2-10
1.
Add 200 mL 10X TBE stock solution to a large graduated cylinder.
2.
Dilute with dH2O to a total volume of 2000 mL.
2 Preparing Gels
March 2001
Applied Biosystems
40% (19:1) Acrylamide Stock Solution
Per 100 mL:
•
Acrylamide
•
Bis-Acrylamide
38.0 g
2.0 g
To prepare the acrylamide solution:
1.
Dissolve the crystalline acrylamide and bis-acrylamide in enough dH2O to
bring the total volume up to 90 mL.
2.
Add 10 g of mixed-bed, ion-exchange resin.
3.
Stir at room temperature until all crystals dissolve, then continue to stir for
an additional 5-10 minutes.
4.
Filter through a 0.2-µm cellulose nitrate cup filter assembly.
5.
Transfer to a 100-mL graduated cylinder and adjust volume to 100 mL with
dH2O.
The finished solution will be 19:1, acrylamide:bis-acrylamide. Solutions are stable
for approximately 1 month when refrigerated at 4 °C.
Reagents and Supplies for Gels
WARNING
CHEMICAL HAZARDS. Urea is a potential mutagen. TEMED is
extremely flammable and can be very destructive to the skin, eyes,
nose, and respiratory system. Do not store, handle, or work with
any chemicals or hazardous materials unless you have received
appropriate safety training and have read and understood all
related Material Safety Data Sheets. Comply with all federal, state,
and local laws related to chemical storage, handling, and disposal.
Ensure that you have each item on the following list ready before beginning to
mix a sequencing gel.
March 2001
•
Urea
•
TEMED
2 Preparing Gels
2-11
Applied Biosystems
•
10% Ammonium persulfate (w/v) in dH2O
Prepare fresh stocks daily, and store refrigerated.
•
Volumetric pipets and tips
•
0.2-µm cellulose nitrate cup filters
•
Plates, spacers, and combs
Optically clear glass plates, spacers, and combs are available from Applied
Biosystems.
2-12
•
Alconox detergent
•
Mixed bed, ion-exchange resin
•
Level gauge and a level working surface
2 Preparing Gels
March 2001
Applied Biosystems
Preparing the Plates (all sizes except 48 cm)
To prepare 48-cm plates, see page 2-22.
Cleaning the Plates
Rinse both sides of the glass plates with warm water, then wash with a detergent
such as Alconox that will not leave a residue. Be careful not to scratch or abrade
the glass surfaces.
Caution
Avoid contacting the glass plates with metal. Scratches caused by
metal objects, such as drying racks or spatulas weaken the glass
and reduce its usable life.
Rinse the plates well with warm water followed by distilled, deionized water. Stand
the plates up and allow them to air dry.
Note
If you are in a hurry, dry the plates gently with lint free towels rather than
with an organic solvent which might contain fluorescent residues.
Fluorescent residues tend to cause a green haze on your gel image.
Notched
plate
Plain plate
Spacers
Figure 2-1. Glass plates for casting gel
March 2001
2 Preparing Gels
2-13
Applied Biosystems
Note
Do not touch the cleaned surfaces of the glass plates and wear gloves
to handle the plates.
Each time you use the glass plates, use them with the same side of each plate on
the inside. After the first use, the front plate has a hydrophobic area where the
buffer chamber gasket made contact with the plate. You must use the front plate
in the same configuration each time to avoid difficulty pouring the gel. You may
mark the outside of each plate the first time you use it. Mark the plates with a tiny
scratch at the top that doesn’t interfere with the read region or the upper buffer
chamber seal.
To clean plates that have been previously used:
This cleaning is usually done immediately after the previous run is complete.
Refer to page 3-47 for instructions.
Applying the Bind-Silane Compound
Note
Bind-Silane use is optional with any of the sharks-tooth combs.
However, it must be used with all square-tooth combs with more than
36 wells.
Silating the unnotched glass plate in the well area (upper 3.5 cm) with a solution
like Pharmacia’s Bind-Silane compound helps eliminate deformed wells. Follow
the instructions below for performing the silation. This must be repeated every
time a new gel is poured. Remove the old gel from the silated area with 0.1 N HCl
or NaOH, or scrape off gently.
To apply the Bind-Silane compound:
2-14
1.
Wash the unnotched glass plate with Alconox detergent, rinse well with
distilled water, and leave to dry.
2.
Adjust 200 mL H2O to pH 3.5 with acetic acid. Add 800 µL Bind-Silane and
stir until clear, approximately 15 minutes.
3.
Set the unnotched plate in a tub with the Bind-Silane solution covering the
top 3.5 cm of the glass. Leave immersed at room temperature for
10–60 minutes.
2 Preparing Gels
March 2001
Applied Biosystems
IMPORTANT
4.
Do not get Bind-Silane on the laser-scanning region of the gel; it will
fluoresce.
Rinse the glass plate with warm water, and then with dH2O, and let dry.
Assembling the Plates
To use your time efficiently, you might want to mix the acrylamide-urea gel
solution and, while it is being stirred, clean and prepare the plates. To mix the
acrylamide-urea solution, refer to page 2-18.
To assemble the plates:
1.
Clean the plates, as described on page 2-13.
2.
Clean the 0.4-mm gel spacers in a similar manner, and make sure they are
the proper length for the glass plates.
If the spacers are longer than the full length of the glass plates, clip them
to the proper size. A paper cutter works well for trimming spacers. The
XL spacers are marked with lines to make cutting easier.
IMPORTANT
Because of tolerance issues, verify the combs, spacers, and casting
comb you are using are compatible (made of the same material) and
are the same thickness.
3.
Place the plain plate on a protected bench top.
4.
Place the gel spacers on the plain plate with the notches at the top of the
plate and facing in.
You may use a pipet to apply small water droplets to the edge of the plate
at several spots along the length so the water flows between the spacers and
the glass to keep the spacers in position.
March 2001
2 Preparing Gels
2-15
Applied Biosystems
Place water droplets here
to hold spacers in position
Spacers
notched
facing in
Spacers
Plain plate
Figure 2-2. Securing spacers on glass plates (top view)
5.
Align the notched plate (inside down) on top of the spacers. The edges of
the glass plates should be flush.
Again, be sure the inside of the plate is free of any particles that might
fluoresce.
6.
Temporarily clamp one side of the plates.
Notched
plate
Plain
plate
Figure 2-3. Plates aligned
2-16
7.
Apply 1.5-inch wide tape tightly along the edge opposite the clamp. Wrap
tape around both the top and bottom corners.
8.
Remove the clamp and apply tape along the second long edge. Wrap tape
around both the top and bottom corners.
9.
Apply tape along the bottom edge of the plates, wrapping around both
corners.
2 Preparing Gels
March 2001
Applied Biosystems
10. Eliminate any air pockets under the tape and reinforce points of assembly
that are likely to leak acrylamide solution.
Note
March 2001
Using a commercially available gel pouring device can simplify gel
casting by eliminating the need to tape the glass plates.
2 Preparing Gels
2-17
Applied Biosystems
Preparing the Gel (all sizes except 48 cm)
Preparing Acrylamide-Urea Gel Solutions
The ABI PRISM 373XL DNA Sequencer gels are very thin. Use only high purity
reagents to prepare them. You can scale the recipe for double or half the amount
as necessary. The recipes can be used for both Sequencing and GeneScan gels.
WARNING
CHEMICAL HAZARD. Urea causes eye, skin, and respiratory
irritation. Lab experiments have shown mutagenic effects. Avoid
contact. Wear chemical resistant gloves, safety goggles, and
other protective clothing.
WARNING
CHEMICAL HAZARD. Acrylamide and bis-acrylamide are
neurotoxins. Avoid inhalation and skin contact. Wear gloves at all
times and work in a fume hood when handling acrylamide
solutions, and use appropriate precautions to avoid inhalation of
crystalline acrylamide.
Preparing 4.25%, 4.75%, or 6% Acrylamide-urea Gel Solutions
Use the following three recipes for all gels except 48 cm gels (for 48-cm gels see
Preparing the Plates (48 cm only) on page 2-22). The volume of 40% acrylamide stock
solution varies between the different gel recipes, and the resulting water needed
to adjust to the total volume differ; everything else remains the same.
1.
Combine the following components in a 150-mL beaker to prepare a final
volume of 80 mL of one of the three gel solutions:
• 40 g urea
• 27 mL dH2O
• 1 g (less for smaller volumes) mixed-bed, ion-exchange resin
2-18
2 Preparing Gels
March 2001
Applied Biosystems
2.
Customize the gel solution by adding the appropriate amount of 40%
acrylamide stock, as follows:
• For a 4.25% acrylamide, 8.3 M urea, 1X TBE (34 cm BaseSprinter) add
8.5 mL 40% acrylamide stock solution.
• For a 4.75% acrylamide, 8.3 M urea, 1X TBE (24 cm BaseSprinter or
34 cm Full or XL Scan) add 9.5 mL 40% acrylamide stock solution.
• For a 6% acrylamide, 8.3 M urea, 1X TBE (12 cm and 24 cm Full or
XL Scan) add 12 mL 40% acrylamide stock solution.
3.
Stir the solution while gently warming it until the urea crystals begin to
dissolve and the flask is slightly warm to the touch. Continue to stir the
solution with no additional heating for 5-10 minutes longer, or until all the
urea crystals have dissolved.
While the solution is being stirred, you can prepare the plates, if you have
not already done so.
4.
Filter the acrylamide solution through a 0.2 µm cellulose nitrate filter,
de-gas for 5 minutes, and transfer to a 100-mL graduated cylinder.
Note
5.
The degas time should be constant for all gels, to ensure a reproducible
polymerization rate for all runs.
Add 8 mL filtered 10X TBE buffer and adjust the volume to 80 mL with
dH2O, if necessary.
IMPORTANT
March 2001
Never add the TBE buffer before removing the mixed bed,
ion-exchange resin by filtration.
2 Preparing Gels
2-19
Applied Biosystems
Casting the Gel (all sizes except 48 cm)
To cast 48-cm gels, see page 2-26.
1.
Gently pour the acrylamide solution into a 150 mL beaker.
2.
Add 400 µL 10% ammonium persulfate (freshly made) and gently swirl.
Avoid adding air bubbles.
3.
Add 45 µL TEMED and gently swirl. Avoid adding air bubbles.
4.
Immediately and carefully cast the gel by holding the plates at an angle to
the bench top, and resting the lip of the solution vessel against the edge of
the notched plate. Pour the solution between the plates and fill to about
3–5 cm from the top edge of the notched plate (the level of gel solution
must be high enough so that the “ears” of the plates are filled when the
plates are laying flat). The solution can also be dispensed using a 60 mL
plastic disposable syringe.
5.
Allow all air bubbles to rise. Gently tap the plates to help trapped bubbles
rise to the surface.
6.
For sharks-tooth combs: Insert the gel casting comb. Wet the gel casting comb
with the acrylamide solution. Be sure there are no bubbles trapped at the
gel solution/casting comb interface.
For square-tooth combs: Insert the square-tooth comb. Be sure there are no
bubbles trapped at the gel solution/comb interface.
2-20
IMPORTANT
Because of tolerance issues, verify the combs, spacers, and casting
comb you are using are compatible (made of the same material) and
are the same thickness.
Note
Ensure that the casting comb is completely dry before wetting it with
acrylamide. Using a wet casting comb can make the surface of the
acrylamide wavy, which can change the way samples migrate through
the gel.
2 Preparing Gels
March 2001
Applied Biosystems
IMPORTANT
7.
Lay the plates in the horizontal position and secure the casting or
square-tooth comb with three large binder clamps. Place the clamps
directly over the region of the gel casting comb that is sandwiched between
the glass plates.
IMPORTANT
8.
Be sure there are no air bubbles trapped at the gel solution/comb
interface after the comb is inserted. Bubbles result in depressions on
the well surface, leading to sample leakage (for sharks-tooth combs)
and/or decreased resolution in affected lanes.
Use a level gauge to ensure that the plates are on a level surface while
the gel is polymerizing.
Wait at least two hours to ensure complete polymerization prior to using
the gel.
For sequencing and GeneScan runs, refer to Section 3 and Section 4,
respectively, the Step-by-Step procedure, for details on how to prepare and
place the gel and glass plates on the instrument.
March 2001
2 Preparing Gels
2-21
Applied Biosystems
Preparing the Plates (48 cm only)
The 48 cm run uses a different gel recipe and set up procedures than any of the
other runs. The following section provides the necessary information for
preparing the gel solution, preparing and setting up the glass plates, and pouring
the gel.
Preparing Gel Casting Equipment for a 48 cm Run
Rinse both sides of the notched and plain glass plates with warm water. Then wash
the plates with a detergent such as Alconox that will not leave a residue and warm
water. Take care not to scratch or abrade the glass surfaces.
Caution
Avoid contacting the glass plates with metal. Scratches caused by
metal objects, such as clamps, drying racks, or spatulas cannot
be detected by eye and will weaken the glass and reduce its
usable life.
Rinse the plates well with warm water followed by distilled, deionized water. Stand
the plates up and allow them to air dry.
Note
Do not touch the cleaned surfaces of the glass plates and wear gloves
to handle the plates. Always try to use the glass plates with the same
side of each plate on the inside.
Preparing Glass Plates for 48 cm Gels
To achieve the high resolution of a 48 cm run, a very flat well surface is required.
This is more difficult with a 4.0% acrylamide gel, which is less rigid than gels with
higher percentages. Silating the unnotched glass plate in the well area (upper
3.5 cm) with a solution like Pharmacia’s Bind-Silane compound will help to
ensure a flat well surface. Follow the instructions below for performing the
silation. This must be repeated every time a new gel is poured. Remove the old gel
from the silated area with 0.1 N HCl or NaOH, or scrape off gently.
2-22
2 Preparing Gels
March 2001
Applied Biosystems
Applying the Bind-Silane Compound
1.
Wash the unnotched glass plate in detergent, rinse well with distilled water,
and leave to dry.
2.
Adjust 200 mL H2O to pH 3.5 with acetic acid. Add 800-µL Bind-Silane and
stir until clear, approximately 15 minutes.
3.
Set the unnotched plate in a tub with the Bind-Silane solution covering the
top 3.5 cm of the glass. Leave immersed at room temperature for
10–60 minutes.
4.
Rinse the glass plate with warm water, and then with dH2O, and let dry.
IMPORTANT
Do not get Bind-Silane on the laser-scanning region of the gel; it will
fluoresce.
Assembling the Glass Plates for a 48 cm Run
1.
Make sure to use the gel spacers and gel casting comb labeled 0.3 mm.
Wash the comb and spacers with warm water, and then with dH2O, and
then with reagent quality ethanol, and let dry.
IMPORTANT
March 2001
The spacers used for 48-cm WTR gels have notches cut out of one end.
When assembling the plates, ensure that the notches are at the top of
the plates, and facing in, towards the well.
2.
Place the unnotched plate on a protected benchtop and arrange the
spacers on the outer edges of the plate. Lay the notched plate on top of the
unnotched plate and spacers. Align the plates and spacers and clamp one
side to temporarily hold plates in position.
3.
Apply 1.5-inch wide tape tightly along the edge opposite the clamp. Wrap
tape around both the top and bottom corners.
4.
Remove the clamp and apply tape along the second long edge. Wrap tape
around both the top and bottom corners.
2 Preparing Gels
2-23
Applied Biosystems
5.
Note
2-24
Apply tape along the bottom edge of the plates, wrapping around both
corners.
Using a commercially available gel pouring device can simplify gel
casting by eliminating the need to tape the glass plates.
6.
Eliminate any air pockets under the tape and reinforce points of assembly
that are likely to leak acrylamide solution.
7.
Place three binder clips over each spacer (on top of the tape), spaced
evenly between the top and bottom of the plates. Be careful to keep the
clips over the spacer and not in the region where the acrylamide will be
poured. Although using binder clips is not recommended with the shorter
gel plates, it does help keep the gel thickness uniform with the longer glass
plates.
2 Preparing Gels
March 2001
Applied Biosystems
Preparing the Gel (48 cm only)
Preparing 4% Acrylamide-urea Gel Solutions
The 48-cm plates hold a larger volume than the other plates. Therefore, this
recipe is for 100 mL and the volume of TBE, APS, and TEMED are greater than
for the other gels also.
To prepare 4% gel solutions:
1.
Combine the following components in a 150 mL beaker to prepare a final
volume of 100 mL of a 4.0% acrylamide, 8.3 M urea, 1X TBE:
• 50 g urea
• 37 mL dH2O
• 10 mL 40% acrylamide stock solution
• 1 g mixed bed, ion-exchange resin
2.
Stir the solution while gently warming it until the urea crystals begin to
dissolve and the flask is slightly warm to the touch. Continue to stir the
solution with no additional heating for 5-10 minutes longer, or until all of
the urea crystals have dissolved.
3.
Filter the acrylamide solution through a 0.2 µm cellulose nitrate filter,
de-gas for 5 minutes, and transfer to a 100-mL graduated cylinder.
Note
4.
The degas time should be constant for all gels, to ensure a reproducible
polymerization rate for all runs.
Add 10 mL 10X TBE, and bring to 100 mL with dH2O, if necessary.
IMPORTANT
March 2001
Never add the TBE buffer before removing the mixed bed,
ion-exchange resin by filtration.
2 Preparing Gels
2-25
Applied Biosystems
Casting the 48-cm Gel
To cast the 48-cm gel:
1.
Gently pour the acrylamide solution into a 250-mL beaker.
2.
Add 500 µL 10% ammonium persulfate (freshly made) and gently swirl.
Avoid adding air bubbles.
3.
Add 50 µL TEMED and gently swirl. Avoid adding air bubbles.
Note
4.
Adjust the volume of TEMED as needed to ensure complete
polymerization in 5 to 10 minutes.
Holding the plates at an angle, immediately and carefully pour the
solution between the plates and fill to about 3–5 cm from the top edge of
the notched plate.
The level of gel solution must be high enough so that the “ears” of the
plates are filled when the plates are laying flat. You can also use a plastic
disposable syringe to dispense the solution.
IMPORTANT
5.
Pour the solution very carefully. It is difficult to remove air bubbles out
from between the longer plates, and any air bubbles can significantly
reduce resolution. Small air bubbles are difficult to see.
Allow all air bubbles to rise. Gently tap the plates to help trapped bubbles
rise to the surface.
Avoid tapping plates in the laser scan region with a hard object. Scratches
or nicks on the plate surface can interfere with data collection.
2-26
6.
Wet the 0.3-mm gel casting comb with the acrylamide solution and insert
it between the two glass plates.
Note
Ensure that the casting comb is completely dry before wetting it with
acrylamide. Using a wet casting comb can make the surface of the
acrylamide wavy and change the way samples migrate through the gel.
2 Preparing Gels
March 2001
Applied Biosystems
IMPORTANT
7.
Lay the plates in the horizontal position and secure the comb with three
large binder clamps. Place the clamps directly over the region of the gel
casting comb that is sandwiched between the glass plates.
IMPORTANT
March 2001
Be sure there are no air bubbles between the glass plates and the
acrylic casting comb after the casting comb has been inserted into the
gel. Bubbles will be wicked into the acrylamide and result in
depressions on the well surface, leading to decreased resolution in
affected lanes.
Use a level gauge to ensure that the plates are on a level surface while
the gel is polymerizing.
2 Preparing Gels
2-27
200 I'M INVISIBLE
Applied Biosystems
3 Using the Instrument in Sequencing Mode
Contents
Introduction
Before You Set Up the Instrument
Preparing Samples
Setting up the Software
Setting Up the Instrument
Preparing the Gel for Loading
Reagents and supplies needed for preparing the glass plates
Preparing the Electrophoresis Chamber
Model-Specific Notation
Automatic Analysis
Setting Up the Software
Setting up the Analysis Software
Starting the Data Collection Software
Creating User-Specific Module Files
Setting Default Parameters (Preferences)
Creating the Sample Sheet
Creating a Run File
Checking the Plates
Cleaning the Plates or Skipping Lanes
Skipping Lanes
Check the PMT Gain Setting
Verify Run Mode
First Time Run
Inserting the Sharks-tooth Comb
Completing Instrument Setup
Preparing and Loading Samples
Pre-running the Gel
Resuspending the DNA
Loading the Samples and Starting the Run
Observing Instrument Status and Data
March 2001
3 Using the Instrument in Sequencing Mode
4
5
5
5
6
6
6
8
8
15
16
16
17
17
19
21
22
27
28
29
32
32
32
34
35
39
39
40
41
45
3-1
Applied Biosystems
Data Collection Status Windows
Real-time Data Windows
Cleaning Up After the Run
Removing the Gel from the Instrument
Cleaning the Plates, Comb, and Spacers
Analyzing the Data
Color of Data Display and Output
Reading an Electropherogram
Archiving the Data
Files Created During a Run
Run Files
Gel Files
Run Log Files
Sample files
.Seq files
Other Files
Sample Sheet Files
Custom Analysis Settings and Instrument Files
3-2
3 Using the Instrument in Sequencing Mode
45
45
47
47
48
50
50
51
52
52
52
52
52
53
53
53
53
53
March 2001
Applied Biosystems
Prepare and label samples
Pour gel
Polymerize
~2 hr
Restart Macintosh
Set up software
Load gel on instrument
Run plate check
Cancel plate check
Add buffer, Start pre-run
Pre-run
~5 min
Run samples
into gel
~2 min
Resuspend DNA
Pause pre-run
Load first set of samples
Resume pre-run
Pause pre-run
Load second set of samples
Cancel pre-run
Start Run
Run
~7–16 hr
Observe status
Analyze and print (automatic)
Clean up
Figure 3-1. Flow chart for electrophoresing/collecting data on ABI PRISM 373 DNA
Sequencer with XL Upgrade
March 2001
3 Using the Instrument in Sequencing Mode
3-3
Applied Biosystems
Introduction
This section guides you through the process of electrophoresing your DNA
samples and collecting the resulting sequence data on the ABI PRISM 373XL DNA
Sequencer. Figure 3-1 shows the recommended flow of the process.
Although this section is detailed, it does not provide complete details for each
procedure. Use it as a guide, and refer as necessary to the cross-referenced pages
for more detail.
Note
Even if you have previous experience using the Model 373, you should
read through these steps. The data collection software has been
streamlined. You no longer need to enter any commands on the
instrument itself.
The information in this section applies to Sequencing applications. Instrument
setup consists of the same steps for either Sequencing or GeneScan applications.
The only differences are in completing the Sample Sheet and Run file.
For further information about the analysis performed for each application, refer
to the Sequencing and GeneScan analysis software manuals.
3-4
3 Using the Instrument in Sequencing Mode
March 2001
Applied Biosystems
Before You Set Up the Instrument
Preparing Samples
Make sure you have prepared your samples as described in the ABI PRISM DNA
Sequencing Chemistry Guide, or in the appropriate kit protocol. Before you set up
your instrument, perform the following procedures:
•
Purify and quantitate the template DNA.
•
Complete the appropriate sequencing reactions with dye primer or dye
terminator chemistry.
•
Prepare a polyacrylamide gel.
It is very important that you purify the DNA thoroughly and have a sufficient
quantity of DNA to get the best results. Optimal quantities for single-stranded and
double-stranded templates are shown in the chemistry guides and protocol
manuals for appropriate kits.
Setting up the Software
While the gel is polymerizing, you can set up the Sample Sheet and Run file for
the data collection software. Refer to Setting Up the Software on page 3-16.
If you want automatic analysis after data collection, set up the conditions that
control data analysis before you start a run. To do so, refer to the user’s manual
provided with your analysis software.
IMPORTANT
If you choose automatic analysis and want the Sequencing Analysis
software to use a setting other than the default, customize the analysis
preferences before you start data collection.
If you specify automatic analysis in the data collection program, the analysis
program starts automatically at the end of the run to process the collected data.
March 2001
3 Using the Instrument in Sequencing Mode
3-5
Applied Biosystems
Setting Up the Instrument
Pour your gel as described in Section 2, and allow it to polymerize for two hours
before proceeding with this section.
IMPORTANT
Make sure to use the appropriate gel concentration for the gel length
and run type.
Preparing the Gel for Loading
Note
Wear clean laboratory gloves throughout this procedure both for your
protection, and to avoid transferring fluorescent contaminants from
your hands to the glass plates.
Reagents and supplies needed for preparing the glass plates
3-6
•
A single-edged razor blade or a second casting comb
•
1.5 L 1X Tris Borate EDTA buffer (2 L for 34-cm gels), pH 8.3 (from 10X
TBE stock solution, Section 2)
•
A clean, 10–50 mL syringe and 22 G needle
3 Using the Instrument in Sequencing Mode
March 2001
Applied Biosystems
To prepare the plates for loading on the instrument:
Figure 3-2. Casting comb
1.
Remove the clamps on the gel casting (single-well) comb, and the tape
from the edges of the plates.
2.
Carefully remove the gel casting comb from between the plates. Remove
any acrylamide in the well region with the razor blade.
Note
3.
Note
March 2001
To avoid cutting the comb, do not use the razor blade while the casting
comb is in place.
Rinse the glass plates with cold water.
a.
Rinse both sides and edges of the glass plates thoroughly with cold tap
water to remove dried acrylamide and urea.
b.
Rinse the well and plates with dH20 and allow the plates to air dry.
Wash the gel casting comb and allow it to air dry, so it will be ready
when you pour the next gel.
3 Using the Instrument in Sequencing Mode
3-7
Applied Biosystems
Preparing the Electrophoresis Chamber
WARNING
ELECTRICAL SHOCK HAZARD. The Model 373 contains a high
voltage power supply. This instrument has been designed with
safety features in the door to disconnect the power supply when
the door is open. Although it is designed for safe operation, please
follow procedures as prescribed. As with any electrophoresis
apparatus, care should be taken during instrument operation and
when handling electrodes and liquids.
WARNING
ELECTRICAL SHOCK HAZARD. Wait at least 5 seconds after
opening the door before touching the electrodes in the
electrophoresis chamber. Capacitors hold an electrical charge
even after the power supply is disconnected.
Model-Specific Notation
This manual applies to three configurations of Model 373 DNA Sequencers:
•
Classic. This configuration includes 370s with Macintosh upgrade and the
373A. Classic models have electrophoresis chambers which support 24-cm
WTR distances.
•
Leon. This configuration has an electrophoresis chamber which supports
6-, 12-, and 24-cm WTR distances.
•
Stretch. This configuration supports 6-, 12-, 24-, 34-, and 48-cm WTR
distances and is easily identified by its extended door.
Note
All three instrument configurations must have five-filter wheel
capability.
Most of the information in this manual applies to all three configurations,
however, some sections of the manual apply to only one or two of these
configurations. These sections are preceded by the name of the configuration
(Classic, Leon, or Stretch) to which they apply.
3-8
3 Using the Instrument in Sequencing Mode
March 2001
Applied Biosystems
To prepare the electrophoresis chamber:
Classic
1.
Open the door to the Model 373 and make sure the heating plate is dry.
2.
Place the lower buffer chamber in the bottom of the electrophoresis
chamber with the electrode to the front. The upper chamber will not be
needed until later.
Upper buffer
chamber
Glass plates
Beam-stop bar
Model 373 (Classic),
side view
Lower buffer
chamber
Figure 3-3. Placement of the Buffer Chambers, glass plates, and beam-stop
bar in the electrophoresis chamber of the Model 373 (Classic)
3.
March 2001
Pull the black beam-stop bar in the electrophoresis chamber toward you
until it stops (Figure 3-3). Rest the bottom edge of the plates on the
notched supports in the bottom of the lower buffer chamber, with the
notched plate facing away from you. Stand the plates in a vertical position.
Press them firmly against the heating plate, and then push the beam-stop
bar up against the plates. Center the plates in relation to the beam-stop bar,
and then lock the beam stop bar in place by sliding the latches down along
the outer edges.
IMPORTANT
Center the plates to ensure that each sample migrates within the read
region of the laser.
Note
You can remove the beam-stop bar. In normal operation you do not
need to remove it. To re-install the beam-stop bar, line up the alignment
pins in the alignment grooves and slide the beam-stop bar into place.
3 Using the Instrument in Sequencing Mode
3-9
Applied Biosystems
Leon
To prepare the electrophoresis chamber:
1.
Open the door to the Model 373.
Upper buffer chamber
support
Heat plate reset
Beam-stop bar
Beam-stop
swivel clamps
Glass plate support
Lower buffer chamber
Figure 3-4. Model 373 (Leon) Electrophoresis Chamber
2.
3-10
Place the lower buffer chamber in the bottom of the electrophoresis
chamber with the electrode to the front. The upper chamber will not be
needed until later.
3 Using the Instrument in Sequencing Mode
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Applied Biosystems
3.
Adjust the upper buffer chamber supports to 24 cm. Loosen the screws and
slide the brackets to the correct position as indicated by the well-to-read
length printed on the supports, and then tighten the screws.
Note
You must use an acrylamide gel with a 24-cm well-to-read distance for
sequencing on the Model 373.
4.
March 2001
Place the glass plates into the instrument as follows:
a.
Pull the black beam-stop bar in the electrophoresis chamber towards
you until it stops (Figure 3-4 on page 3-10).
b.
Rest the bottom edge of the plates on the notched supports in the
bottom of the lower buffer chamber, with the notched plate facing
away from you.
c.
Stand the plates up in the vertical position. Press them firmly against
the back of the chamber (avoid touching the laser read region, and
then push the beam-stop bar up against the plates.
d.
Center the plates with relation to the beam-stop bar and then latch
the bar in place.
3 Using the Instrument in Sequencing Mode
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Applied Biosystems
To Set Up the Electrophoresis Chamber:
Stretch
1.
Open the door to the Model 373. The electrophoresis chamber is shown in
Figure 3-5.
48-cm WTR
riser shelf
34
-cm
WT
R
Upper buffer chamber
supports and shelf
24
-cm
WT
R
Beam-stop bar
Gel plate rests
(ABS block)
Lower buffer chamber
Figure 3-5. Model 373 (Stretch) Electrophoresis Chamber
2.
3-12
For 24- or 34-cm gel: place the straight white upper buffer chamber shelf on
the appropriate support on the inside of the electrophoresis chamber (see
Figure 3-5).
3 Using the Instrument in Sequencing Mode
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Applied Biosystems
For 48-cm gel: Place the straight white plastic upper chamber shelf on the
34-cm support. Place the 48-cm WTR extension shelf on top of the straight
shelf. The upper buffer chamber is placed on this shelf.
3.
Slide the heat plate into place, as follows:
a.
Choose the correct heat plate for the glass plates you are using.
b.
Make sure the electrical wires for the temperature sensor are at the
top, with the plate clamps toward the front. Align the grooves on
either side of the heat plate with the heat plate guides on the
chamber.
c.
Slide the heat plate down until it rests on the bottom of the chamber.
d.
Plug the temperature sensor on the heat plate into the socket at the
top left of the chamber.
e.
Plug the heat plate ground into the socket at the top right of the
chamber.
IMPORTANT
4.
Make sure the heat plate ground is connected before continuing.
Adjust the gel plate rests in the lower buffer chamber, if necessary (see
Figure 3-6). The gel plates rests are located in the bottom of the buffer
chamber and serve to elevate the glass plates. The 34-cm plates must sit
higher in the buffer chamber to rest in the correct position. All other
plates rest in the lower position.
Each gel plate rest consists of several parts, a large ABS block on the
bottom, a shorter ABS block on top of it, and an adjustable thumb screw.
When the thumb screw is loose, the small block can be moved forward and
back, so that the plates rest either on one block, or on two blocks (see
Figure 3-6).
March 2001
a.
Loosen the thumb screws.
b.
Slide the blocks toward the back of the chamber if you are using
34-cm plates, so the plates rest in the high position. Slide the blocks
toward the front of the chamber for all other gel lengths so the plates
rest in the low position.
c.
Tighten the thumb screws.
3 Using the Instrument in Sequencing Mode
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Applied Biosystems
Thumb screw
a. For all run lengths except 34 cm. With
the movable blocks toward the front of the
electrophoresis chamber, the glass plates rest
lower in the buffer chamber.
Front of electrophoresis chamber
Glass plates rest here
Thumb screw
b. For 34-cm runs only. With the movable
blocks toward the back of the electrophoresis
chamber, the glass plates rest higher in the
buffer chamber.
Front of electrophoresis chamber
Figure 3-6. Adjustable gel plate rests
3-14
5.
Place the lower buffer chamber in the bottom of the electrophoresis
chamber with the electrode to the front. The upper chamber will not be
needed until later.
6.
Unfasten the latches on either side of the beam-stop bar and swing the
beam-stop bar forward, toward you.
7.
Place the glass plates in the electrophoresis chamber in front of the heat
plate and read window, with the bottom of the plates resting on the gel
plate rests.
3 Using the Instrument in Sequencing Mode
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Applied Biosystems
a.
Center the glass plates carefully, or the beam-stop bar will not fasten.
b.
Press the glass plates against the heat plate and close the beam-stop
bar.
c.
Fasten the latches at either side of the beam-stop bar.
IMPORTANT
Do not touch the gel plates within the read region.
Automatic Analysis
If you want automatic analysis after data collection, set up the conditions that
control data analysis before you start a run. To do so, refer to the user’s manual
provided with your analysis software.
IMPORTANT
If you choose Automatic Analysis and want the Sequencing Analysis
software to use settings other than the default, customize the analysis
preferences before you start data collection. Define all preferences
from the Edit pulldown menu.
If you specify automatic analysis in the data collection program, the analysis
program starts automatically at the end of the run to process the collected data.
March 2001
3 Using the Instrument in Sequencing Mode
3-15
Applied Biosystems
Setting Up the Software
Before starting electrophoresis, ensure there is sufficient space on the hard disk
and restart the Macintosh. After deleting files, you must restart the Macintosh.
You must also set up certain files in the data collection software:
•
Set preferences
•
Record sample information in the Sequence Sample Sheet
•
Create a Sequence Run file
Note
These tasks can be performed while you are waiting for the gel to
polymerize.
You can simplify creation of the files by setting preferences for certain parameters
in each file.
The procedures described here do not include detailed descriptions of the data
collection software. They are intended to give you a guide for steps you perform
and the order in which to perform them. For specific information about the
software and how to complete specific fields, refer to Section 5.
You may not need to change these default parameters for future runs, if you always
run the same or similar parameters. However, it is important to verify these
defaults, since they determine the names and locations of your data.
For more detail, refer to the specific pages in Section 5.
Setting up the Analysis Software
If you want automatic analysis after data collection, set up the conditions that
control data analysis before you start a run. To do so, refer to the User’s Manual
provided with your analysis software.
IMPORTANT
3-16
The default Base Caller used by the Sequencing Analysis software is
SemiAdaptive. If you want to use the ABI BaseCallers or Adaptive Base
Caller, change the analysis preferences before you start data
collection.
3 Using the Instrument in Sequencing Mode
March 2001
Applied Biosystems
Starting the Data Collection Software
1.
Choose Restart from the Special menu in the Finder to restart your
Macintosh.
2.
The data collection software should automatically open upon restarting
the Macintosh. If it does not open, double-click the icon to open the
program. Refer to To make the data collection program open at startup: on page
5-13 for instructions to open the collection program at startup.
Note
When you start the ABI PRISM 373 DNA Sequencer with XL Upgrade
data collection program, the program searches for the current version
of firmware on the Translation Interface Processor (TIP). If none is
found, the program automatically copies the firmware image file (stored
on the Macintosh) to the TIP. If the TIP’s version of firmware is different
from the collection software version, a dialog box appears, allowing you
to continue or send a new firmware to the TIP. Click Send New
Firmware to update your firmware version.
Creating User-Specific Module Files
The ABI PRISM 373 DNA Sequencer with XL Upgrade is shipped with module file
templates for plate check, prerun, sequencing, and GeneScan runs. Table 3-1
shows the default parameters for the module file templates.
IMPORTANT
The parameters in Table 3-1 apply only to 24-cm WTR Classic or Leon
6% acrylamide gels.
Table 3-1. Module File Default Parameters
Module
Plate Check
Pre Run
Seq Run
GS Run
March 2001
Field
(Volts)
-2500
2500
2500
Power
(Watts)
-30
30
30
Current
(mAmps)
-40
40
40
Laser Power
(mWatts)
40
40
40
40
3 Using the Instrument in Sequencing Mode
Run Time
(Hours)
0.50
0.33
12
12
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Applied Biosystems
You must create user-specific module files the first time the instrument is used and
for each different run condition. Once you modify a module file template, you
can save it and use it each time a similar run is performed. Refer to Table 1-2 on
page 1-14 and Appendix B for suggested module file and Run parameters.
To modify a module file template:
1.
Choose New from the File menu.
2.
Click the Sequence Run or GeneScan icon in the window that appears.
3.
Choose the module file you wish to modify from the Run Module pop-up
menu.
Note
You do not need to complete the other information in the Run window
to modify a module file.
4.
Click the small document icon next to the Run module pop-up menu.
Figure 3-7. Module settings dialog box
5.
Type a new number in the applicable entry fields. Refer to Table 1-2 on
page 1-14 for recommended run parameters.
6.
Save the changes.
• Save as Default: To save the changed parameters as defaults for the
module without changing the module name, click Save as Default. The
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3 Using the Instrument in Sequencing Mode
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Applied Biosystems
new settings are permanently saved and the name of the module file
remains unchanged.
• Save Copy in…: To save the changed parameters as defaults, click Save
Copy in. Type in a new name for the module file and save the file in the
Modules Folder. The new settings are permanently saved and the name
of the module file is changed.
Note
You will not be able to select your new module file in the pop-up menu
until you save and close the Run window, then reopen the saved Run
file or create a new Run file.
• Save: To use the changes as a one-time override for the current run,
click Save.
Note
Custom module files are stored on your hard disk and can be used for
any subsequent run. If you reload your collection software, however,
you must re-create your custom modules.
Setting Default Parameters (Preferences)
You might only need to set default parameters prior to the first run. However, if
more than one person uses the instrument, we recommend you set or verify all
preferences before starting the run. If you are satisfied with the defaults stored in
the instrument, skip to page 3-21 to create a Sample Sheet and a Run file.
You can set several types of preferred defaults:
March 2001
•
Folder locations: The ABI PRISM 373XL data collection software uses
specified folders for data storage. This option allows you to specify the
folders it uses. For information about changing folder locations, refer to To
change one of the data storage folders: on page 5-17.
•
Default File names: The data collection program automatically names new
files and folders created during a run. You can specify the prefix and suffix
of each type of file (see Setting File Name Preferences on page 5-18).
•
Sequence Sample Sheet defaults: You can set defaults for certain parameters on
the Sample Sheet. Since the Sample Sheets differ for Sequence Analysis
3 Using the Instrument in Sequencing Mode
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Applied Biosystems
and GeneScan (fragment) Analysis, the parameters for which you can set
defaults also differ. To set defaults for the sequence Sample Sheet, refer to
To set Sample Sheet defaults for Sequencing Analysis applications: on page 5-21 .
•
Sequence Run defaults: You can also set defaults for certain parameters in the
Run file. As with the Sample Sheet defaults, separate defaults apply for
sequence runs and GeneScan analysis runs. To set defaults for sequence
Run files, refer to Setting Defaults for Sequencing Analysis Applications on page
5-21.
•
General settings: The ABI PRISM 373XL instrument is normally attached to
the Modem port of the Macintosh computer. If you need to change the
connection, you can specify the change using this preference choice. Also
use this choice to reset the global serial number (refer to General Settings on
page 5-27). To change general settings, refer to To change the general settings:
on page 5-28.
•
Dye indicators: Each of the four dyes used during a run has a preset code and
color. You can change the color assigned to each dye, and change the color
that appears in the plot when it is printed. To do so, refer to To set dye
indicator preferences for the analysis applications: on page 5-29.
To set preferences:
1.
Choose Preferences from the Window menu and choose the appropriate
option from the submenu that appears.
Figure 3-8. Preferences submenu
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3 Using the Instrument in Sequencing Mode
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Applied Biosystems
2.
Enter or change the defaults, as described on the pages noted above.
3.
Click OK to accept the changes.
Creating the Sample Sheet
Before starting a run you must record sample information in the Sequence
Sample Sheet and the Sequence Run file. Once you have set up the Sample Sheet,
you can automatically import the Sample Sheet information into the Run file,
which associates sample information (name and type of analysis) with each lane
position in the gel.
Separate Sample Sheets exist for sequencing and GeneScan analysis applications
(see Figure 3-9). Be sure to choose Sequence Sample Sheet.
If a Sample Sheet containing the correct information already exists, you can use
it. In most cases, you need to create a new Sample Sheet for each run. You can
enter the information directly or export information from a database in
tab-delimited format and import it into the Sample Sheet.
To create a Sample Sheet:
IMPORTANT
Although the Sample Sheet has 72 lanes available, fill in the sample
information only for the number of lanes you are using.
1.
Choose New (z-N) from the File menu.
2.
Click the Sequence Sample Sheet icon in the window that appears.
IMPORTANT
Do not mix sequence analysis and GeneScan analysis samples on the
same Sample Sheet or the same Run.
The Sample Sheet is similar to a spreadsheet or ledger. It accepts more text
than fits in the visible fields and automatically scrolls as you type your entry.
To view the beginning of a long entry, use the keyboard arrow keys. Many
of the cells have default values that are automatically filled in for each
sample.
March 2001
3 Using the Instrument in Sequencing Mode
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Applied Biosystems
3.
Enter the necessary information. For details about completing the Sample
Sheets (including information about importing from a database), refer to
Creating and Editing a Sample Sheet on page 5-32.
4.
When you are finished, choose Save from the File menu.
The dialog box that appears shows the default file name and storage
location as defined in the File Names and Folders locations preferences,
respectively. You can change the file name if you wish.
5.
Click Save.
6.
Choose Close from the File menu, or click the close box.
Click an arrow to display a pop-up menu of options
Click a title
to select the
entire column
Enter Sample
name here.
When you set defaults for mobility and matrix files, the columns are filled in automatically
Figure 3-9. Blank Sequence Sample Sheet
Creating a Run File
In the Run file, you specify the lanes in which the samples will run and parameters
to be used for both the pre-run and the run. If a Run file exists that contains the
proper parameters, you can copy it to a new folder and use it. In most cases you
need to create a Run file for each new run.
Separate Run windows exist for sequencing and GeneScan analysis applications.
They are very similar; the GeneScan analysis version has an additional (Analysis
Settings) column. Be sure to choose the correct type for your application.
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3 Using the Instrument in Sequencing Mode
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Applied Biosystems
To create a Run file:
1.
Choose New from the File menu.
2.
Click the sequence Run icon in the window that appears.
Small document icons open the selected module or
Sample Sheet for review
Specify length of run
Run control
buttons
Pop-up menus in
these panels allow
you to choose the
modules
(parameters), the
associated
Sample Sheet, the
number of lanes,
the separation
distance (WTR),
and the
instrument file for
the run
Lane numbers cannot be edited
Select filter set
Figure 3-10. Sequence Run window
3.
Choose the parameters for the run from the pop-up menus.
Note
Any changes made in your preferences will not take effect until you
open a new Run file.
a.
Choose modules to be used for the plate check, the pre-run and the
run in the Module pop-up menus.
b.
Choose the Sample Sheet from the Sample Sheet pop-up menu.
When you choose the Sample Sheet, the information about the lanes
in which the samples are to run is imported automatically.
March 2001
3 Using the Instrument in Sequencing Mode
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Applied Biosystems
IMPORTANT
c.
If you select number of lanes less than the number of lanes you filled
out in the in your Sample Sheet, the information will be truncated. To
fix this:
1. Select correct number of lanes and Run mode.
2. Select <none> on the Sample Sheet pop-up menu and reselect
the Sample Sheet you want to use.
Choose an instrument file from the Instrument File pop-up menu.
Appendix C describes Sequencing Instrument Files.
d.
Choose the number of lanes and the separation distance from the
lanes pop-up menu and the WTR pop-up menu, respectively.
e.
Choose XL Scan, Full Scan, or BaseSprinter in the Run Mode pop-up
menu. Use the table below to determine the appropriate run mode
for the lane configuration you are using.
Lanes
18
24
32
36
48
64
Note
3-24
Comb
Sharks-tooth
Sharks-tooth
Sharks-tooth
Sharks-tooth
Sharks-tooth
Sharks-tooth
Run Mode
BaseSprinter
Full Scan
Full Scan
Full Scan
XL Scan
XL Scan
If you select a run mode which is incompatible with the number of lanes
selected in Step d, the lane pop-up menu will display <none>.
3 Using the Instrument in Sequencing Mode
March 2001
Applied Biosystems
4.
Enter the length of the run in the Collect Time entry field. Following are
some suggested run times.
Plate Length
(cm)
24
24
34
34
48
5.
Type of Run
XLScan or Full Scan
BaseSprinter
XLScan or Full Scan
BaseSprinter
XLScan or Full Scan
Typical Collection Time
(hr)
12
6.5
12
7
16
Enter your name in the Operator entry field.
As noted, the sample information for each lane is entered automatically in
the grid at the bottom of the Run window when you choose a Sample
Sheet.
Note
6.
You cannot edit the Sample Sheet information in the Run window.
Make any changes in the Sample Sheet, save the changes, select
<none> and then select the Sample Sheet again in the Run window to
make the changes effective.
Choose the appropriate filter set:
For Sequenase T7 Terminator chemistry: Select filter set B.
For all other sequencing chemistries: Select filter set A.
IMPORTANT
7.
Failure to select the correct filter set will result in the loss of data.
Select the Auto Analyze and Auto Print checkboxes if you want the analysis
program to analyze and print the data.
When you finish completing the information in the Run file, do not close the Run
file window. You can use it to perform a plate check, as described in the next
section.
The options available to you at this time are:
•
March 2001
Save the file for future use, then click the close box to close it.
3 Using the Instrument in Sequencing Mode
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Applied Biosystems
•
Start a plate check by clicking the Plate Check button.
To save the file, choose Save from the File menu. To perform a plate check, see
page 3-27.
For more information about creating the Run file, refer to page 5-40.
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Applied Biosystems
Checking the Plates
Scan the gel and plates before adding buffer and loading samples, to ensure that
no peaks are produced by fluorescent particles on the glass plates or in the gel.
Click Plate
Check after you
choose the Plate
Check module
Figure 3-11. Completed Sequencing Run window
To check the plates:
1.
Note
Click Plate Check in the Run window.
If a dialog box appears indicating you do not have enough room on your
hard disk to save the files created by a run, delete files (particularly Gel
files) to create enough space, making sure to back them up on a floppy
disk or other storage device first, then click Plate Check again.
The laser scans the plates without electrophoresis to detect any unwanted
fluorescent material in the read region.
2.
March 2001
Observe the Scan window that appears.
3 Using the Instrument in Sequencing Mode
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Applied Biosystems
The Scan window should show a relatively flat line across the screen in each
of the four colors. Typically the lowest line is blue, followed by the green,
yellow (black), and red lines. If the scan line is flat, then the plates are
clean. Proceed to Check the PMT Gain Setting on page 3-32.
If you see peaks in the Scan window, clean the glass plates again, as
described below.
WARNING
LASER HAZARD. Do not look directly into the laser beam or allow
a reflection of the beam to enter your eyes. Exposure to direct or
reflected laser light at 40 mW for 0.1 seconds can burn the retina
of the eye and leave a permanent blind spot.
Cleaning the Plates or Skipping Lanes
Peaks in the scan lines could indicate dirt on the glass or contaminating
fluorescence in the gel. To eliminate dirt on the glass, clean the plates and scan
them again. If peaks still appear and you wish to use the gel, avoid loading samples
in the lanes where the peaks appear.
To clean the plates and re-scan:
1.
Click Pause to pause the plate check.
2.
Open the door of the instrument and unlatch the beam stop bar and
remove the plates.
3.
Carefully re-clean both sides of the glass in the laser read region with dH2O
and a lint-free tissue or ultra-pure pressurized air.
4.
Replace the plates, center, and latch the beam stop bar in the
electrophoresis chamber.
5.
Click Resume and check the plates again.
If the peaks do not disappear after cleaning the glass, the gel mixture or buffer
might contain particles. If you wish to use the gel, avoid loading samples in the
lanes where the peaks appear.
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3 Using the Instrument in Sequencing Mode
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Applied Biosystems
Note
Occasionally, peaks in the scan window are caused by a dirty beam
stop bar, rather than by dust or particulates in the gel. Clean the beam
stop bar with dH2O and (optionally) a thin layer of oil, if the peaks do not
disappear after cleaning the glass.
Skipping Lanes
If, even after cleaning, a plate check shows peaks in some lanes of a gel, use Table
3-2 to determine which lanes are contaminated. Do not load samples in those
lanes.
Table 3-2 shows scan numbers that correspond to the center of each lane for five
different sharks-tooth combs. The exact positions of the channels may vary slightly
from instrument to instrument, and with each comb placement.
If the contaminant peaks are close to the edge of a lane, you may wish to skip two
lanes to ensure that there is no contamination in the lanes you load.
Note
Table 3-2 is based on the premise that all lanes run straight and do not
deviate from the “expected” path, so channel/lane center may be an
approximation, depending on the quality of your gel.
Table 3-2. Channel Numbers at Center of Each Lane for Sharks-tooth Combs
24-well comb
Lane Channel
1
7
2
15
3
23
4
30
5
38
6
46
7
54
8
62
9
69
10
77
11
85
12
93
March 2001
32-well comb
Lane Channel
1
5
2
11
3
17
4
23
5
29
6
35
7
41
8
47
9
53
10
58
11
64
12
70
36-well comb
Lane Channel
1
4
2
9
3
14
4
20
5
25
6
30
7
36
8
41
9
46
10
52
11
57
12
62
48-well comb
Lane Channel
1
7
2
15
3
23
4
30
5
38
6
46
7
54
8
62
9
69
10
77
11
85
12
93
3 Using the Instrument in Sequencing Mode
64-well comb
Lane Channel
1
5
2
11
3
17
4
23
5
25
6
35
7
41
8
47
9
53
10
58
11
64
12
70
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Applied Biosystems
Table 3-2. Channel Numbers at Center of Each Lane for Sharks-tooth Combs
(continued)
24-well comb
Lane Channel
13
101
14
107
15
116
16
124
17
132
18
140
19
148
20
155
21
163
22
171
23
179
24
187
3-30
32-well comb
Lane Channel
13
76
14
82
15
88
16
94
17
100
18
106
19
112
20
117
21
123
22
129
23
135
24
141
25
147
26
153
27
159
28
165
29
171
30
177
31
183
32
189
36-well comb
Lane Channel
13
68
14
73
15
78
16
84
17
89
18
94
19
100
20
105
21
110
22
116
23
121
24
126
25
132
26
137
27
142
28
148
29
153
30
158
31
164
32
169
33
174
34
180
35
185
36
190
48-well comb
Lane Channel
13
101
14
107
15
116
16
124
17
132
18
140
19
148
20
155
21
163
22
171
23
179
24
187
25
195
26
203
27
211
28
218
29
226
30
234
31
242
32
250
33
257
34
265
35
273
36
281
37
289
38
297
39
306
40
314
41
322
42
330
43
338
44
345
45
353
3 Using the Instrument in Sequencing Mode
64-well comb
Lane Channel
13
76
14
82
15
88
16
94
17
100
18
106
19
112
20
117
21
123
22
129
23
135
24
141
25
147
26
153
27
159
28
165
29
171
30
177
31
183
32
189
33
194
34
200
35
206
36
212
37
218
38
224
39
230
40
236
41
242
42
248
43
258
44
264
45
264
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Applied Biosystems
Table 3-2. Channel Numbers at Center of Each Lane for Sharks-tooth Combs
(continued)
24-well comb
32-well comb
36-well comb
48-well comb
Lane Channel Lane Channel Lane Channel Lane Channel
46
361
47
369
48
377
March 2001
3 Using the Instrument in Sequencing Mode
64-well comb
Lane Channel
46
270
47
276
48
282
49
288
50
294
51
300
52
305
53
311
54
317
55
323
56
329
57
335
58
341
59
347
60
353
61
359
62
365
63
371
64
377
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Applied Biosystems
Check the PMT Gain Setting
Verify Run Mode
The PMT gain setting differs for each of the run modes (XL Scan, Full Scan, and
BaseSprinter). Verify that you have selected the correct run mode in the Run
window before checking the PMT gain setting.
First Time Run
The PMT gain settings are stored in a Calibration file. If this is the first time the
instrument is being used, you should make and send a Calibration file. Refer to
Section 5 for instructions on how to make and send a Calibration file.
Recommended PMT Gain
The PMT gain should be set such that the lowest scan line (usually the blue line)
is positioned at approximately 900 on the y-axis in the Scan window. For
subsequent runs, the PMT gain will need to be adjusted only if the y-axis value is
out of the range of 800–1000 on the y-axis. Different PMT gains are required for
the three run modes (XL Scan, Full Scan, and BaseSprinter).
To check the PMT gain:
1.
While the Plate Check is running, view the four base lines (each line
represents one of the four filters) in the Scan window of the 373XL
collection program.
2.
Position the cursor so the tip of the arrow just touches a flat section of the
lowest scan line (usually the blue line), as in Figure 3-12.
Read y-axis value here
Figure 3-12. Cursor positioned correctly to measure PMT gain
3-32
3 Using the Instrument in Sequencing Mode
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Applied Biosystems
3.
Read the y-axis value displayed in the upper-left hand corner of the scan
window.
• If the lowest scan line is positioned between 800–1000 on the y-axis,
cancel the Plate Check, and then proceed to Inserting the Sharks-tooth
Comb.
• If the lowest scan line is outside the recommended range, adjust the
PMT gain.
To adjust the PMT gain:
1.
While the Plate Check is running, choose Manual Control from the
Window menu.
2.
Adjust the size and position of the Scan and Manual Control windows so
you can see both windows without any overlap.
3.
In the Manual Control window, choose PMT Gain from the Fxn Name
pop-up menu.
4.
Type a new value, then click Execute.
Note
If you enter a larger number than the one displayed, the lines in the
Scan window will shift up; a smaller number causes them to shift down.
5.
Check the y-axis value in the Scan window. (First click on the Scan window
to make it active.)
6.
Repeat steps 3–5 until the PMT gain is within the recommended range.
7.
Close the Manual Control window.
8.
Cancel the Plate Check in the Run window.
Note
March 2001
If you select a different run mode, you must change the PMT gain.
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Applied Biosystems
Inserting the Sharks-tooth Comb
IMPORTANT
1.
Because of tolerance issues, be sure that the combs, spacers, and
casting comb you are using are compatible (made of the same material)
and are the same thickness.
Insert the sharks-tooth comb (see Figure 3-13).
a.
Center the sharks-tooth comb in the glass notch and carefully slide it
between the glass plates. Make sure all of the points on the comb
enter the space between the plates at the same time to avoid bending
or breaking the teeth. Carefully align the center registration line with
the registration mark on the plates.
b.
Slide the comb down until the tips of all the teeth just touch or slightly
depress the surface of the gel. Do not deform the wells. Make sure the
teeth just barely indent the well surface. All of the teeth must touch
the surface of the gel. If the surface of the gel is not completely flat in
the loading region, some of the teeth may have to be inserted below
the surface of the gel (up to 0.5 mm) so all of the teeth touch the gel
surface. If a tooth has penetrated the gel surface, do not attempt to
withdraw the comb or sample will leak into adjacent wells.
Figure 3-13. Sharks-tooth comb just touching the gel
3-34
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Applied Biosystems
Completing Instrument Setup
Classic 1.
2.
Clamp the glass plates to the upper buffer chamber by placing the
sharks-tooth alignment brace (the rectangular acrylic brace) against the
front of the glass plates and inside the brackets of the buffer chamber.
3.
Tighten the screws on each end of the alignment brace equally on both
sides to maintain even pressure and to ensure that the brace does not slip
up at one end. Turn the screws until you see the gasket compress
completely against the glass. Finger-tighten only—do not overtighten. You
may need to apply light pressure on the top of the brace to prevent it from
moving during tightening.
Note
The main function of the alignment brace is to maintain a firm, even
pressure on the sharks-tooth comb with the glass plates, to prevent
leakage of the sample around the teeth.
4.
Fill the upper buffer chamber with the 1X TBE buffer prepared earlier to
at least half-way up the notched part of the plate (~ 1.3 cm). Check for
leaks. Close the lid of the buffer chamber.
Note
If there is a leak, tighten the brackets slightly. If the leak continues,
siphon the upper buffer, then clean the gasket area on the chamber and
the area of the plate that contacts the gasket. Reassemble as before.
5.
Note
March 2001
After performing a plate check, place the upper buffer chamber at the top
of the gel plates by placing it on the upper shelf (Figure 3-3 on page 3-9)
and sliding the front brackets of the chamber over the glass plates. The
brackets should sit over the spacers on the outside edges of the plate.
Carefully fill the lower buffer chamber with 1X TBE buffer to about
two-thirds full.
Avoid splashing buffer against the back of the chamber since it will spill
over into the liner. Remove any bubbles that are under the bottom edge
of the gel plates using a clean needle and syringe. Bend the needle at
an angle to guide the bubbles out.
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Applied Biosystems
Leon
6.
Attach the top and bottom electrode leads located on the left side of the
electrophoresis chamber and close the door gently.
1.
After performing a plate check, slide the heat plate into place against the
glass plates, as follows (see Figure 3-4 on page 3-10):
a.
Rest the lower edge of the heat plate (the edge with the supports
protruding on both sides) on the upper inside ledge of the beam-stop
bar where it meets the glass plate. The supports rest in the notches.
b.
Stand the heat plate vertically against the glass plates.
c.
Secure the beam-stop swivel clamps.
2.
Place the upper buffer chamber at the top of the gel plates, on the
supports. Slide the clamps over the plates.
3.
Put the alignment brace in place against the front of the glass plates, inside
the brackets of the buffer chamber, and resting on top of the heat plate.
The alignment brace is made of highly polished acrylic so the numbers and
the comb are readily visible through the plastic. Do not mark the
alignment brace with felt-tipped pens, as the use of most organic solvents
to remove the ink will dull the surface of the brace and reduce its clarity.
Some ink may also fluoresce.
4.
Tighten the screws on both ends of the upper buffer chamber equally to
maintain even pressure across the alignment brace, and to ensure that the
brace does not slip up at one end. Turn the screws just until you see the
gasket compress completely against the glass—do not overtighten. You may
need to apply light pressure on the top of the brace to prevent it from
moving during tightening.
The main function of the alignment brace is to maintain a firm, even
pressure on the sharks-tooth comb with the glass plates to prevent leakage
of the sample around the teeth.
5.
3-36
Fill the upper buffer chamber with the 1X TBE buffer prepared earlier to
at least 2/3 up the notched part of the plate (greater than 1.3 cm). Check
for leaks. Close the lid of the buffer chamber.
3 Using the Instrument in Sequencing Mode
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Applied Biosystems
If there is a leak, slightly tighten the brackets. If the leak continues, siphon
the buffer out of the upper buffer chamber and clean the gasket area on
the chamber. Also, clean the area of the plate that contacts the gasket.
Reassemble as before.
Stretch
6.
Carefully fill the lower buffer chamber with 1X TBE buffer to about
two-thirds full. Avoid splashing buffer against the back of the chamber
because it will spill over into the liner. Bubbles should be removed if they
are under the bottom edge of the gel plates. This can be done with a clean
needle and syringe, with the needle bent at an angle to guide the bubbles
out.
7.
Attach the top and bottom electrode leads located on the left side of the
electrophoresis chamber and gently close the door.
1.
After performing a plate check, place the upper buffer chamber at the top
of the gel plates, on the white plastic shelf. Slide the clamps over the plates
but do not tighten the clamps. To ensure a tight seal between the buffer
chamber and the glass plates, make sure the gasket on the upper buffer
chamber is dry.
IMPORTANT
2.
If you have two different upper buffer chambers, use the larger of the
two for 34-cm and 48-cm runs. Use the small upper buffer chamber for
24-cm runs.
Slide the plain acrylic alignment brace over the comb (the one without the
temperature sensor), between the upper buffer chamber clamps and the
glass plates. Finger-tighten the clamps; be careful not to overtighten.
The alignment brace is made of highly polished plastic so the numbers and
the comb are readily visible through the clear plastic. Do not mark the
alignment brace or glass plates with felt-tipped pens, as the use of most
organic solvents to remove the ink will dull the surface reduce its clarity.
Some ink may also fluoresce.
3.
March 2001
Plug the electrode lead on the upper buffer chamber into one of the two
jacks. For 34-cm and 48-cm gels, use the top (rear) socket, and for all other
gels, use the lower (front) socket.
3 Using the Instrument in Sequencing Mode
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Applied Biosystems
4.
Fill the upper buffer chamber to at least two-thirds up the notched part of
the plate (greater than 1.3 cm). Check for leaks. Close the lid of the buffer
chamber.
If there is a leak, slightly tighten the brackets. If the leak continues, siphon
the buffer out of the upper buffer chamber and clean the gasket area on
the chamber. Also, clean the area of the plate that contacts the gasket.
Reassemble as before.
5.
Carefully fill the lower buffer chamber with 1X TBE buffer to about
two-thirds full. For 34-cm runs, fill the lower buffer chamber until the
bottom of the glass plates is covered. Avoid splashing buffer against the
back of the chamber because it will spill over into the liner. Bubbles should
be removed if they are under the bottom edge of the gel plates. This can
be done with a clean needle and syringe, with the needle bent at an angle
to guide the bubbles out.
IMPORTANT
6.
3-38
The buffer must completely cover the bottom of the gel plates. You will
need to prepare 2 L of buffer for 34-cm and 48-cm gels. For all other
lengths, the standard volume of 1.5 L is sufficient.
Attach bottom electrode lead located on the left side of the electrophoresis
chamber.
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Applied Biosystems
Preparing and Loading Samples
Pre-running the Gel
Pre-run the gel for 5 minutes to make sure all connections and components of the
electrophoresis system are working properly.
While the pre-run is in progress, resuspend the DNA in preparation for loading.
To pre-run the gel:
1.
Flush all of the wells with running buffer. Direct the 22 G needle of a
syringe filled with running buffer at the space between adjacent comb
teeth, and gently flush the well.
Remove any bubbles under the bottom edge of the gel plates in the lower
buffer chamber. Use a clean needle and syringe, with the needle bent at an
angle to guide the bubbles out.
2.
If you have not done so, choose the PreRun module from the PreRun
pop-up menu.
3.
Click the PreRun button in the Run window.
The instrument performs electrophoresis without collecting data during a
pre-run.
March 2001
4.
Pre-run the gel for 5 minutes.
5.
Click Cancel in the Run window.
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Applied Biosystems
Resuspending the DNA
Resuspend the DNA in loading buffer, as described below. After the pre-run
(about 5 minutes), load the sample and start the run. When using 48- and 64-well
combs, you can obtain optimum accuracy by loading 1/4 to 1/8 of the total
resuspension volume, e.g., resuspend your dry sample in 8–12 µL loading buffer
and load only 1.5 µL.
IMPORTANT
Do not overload your lanes. Both volume and quantity excess lead to a
decrease in base calling accuracy. Best results are obtained when the
average signal strengths for G, A, T, and C are all below 500
fluorescent units/base. More signal is no guarantee of better results;
with 48 and 64 wells, it actually translates to less accuracy. You may
need to optimize what volume in this range is best for you.
WARNING
CHEMICAL HAZARD. Formamide is a known teratogen (can cause
birth defects). Before using formamide, read the MSDS found in
the Safety Summary provided previously. Wear protective
eyewear, gloves, and safety clothing when working with
formamide. Wash thoroughly after handling formamide.
To resuspend the DNA:
1.
Resuspend your DNA in loading buffer (5:1 deionized formamide:
25–50 mM EDTA with 25–50 mg/mL Blue Dextran).
Use the following table to determine the appropriate resuspension and
loading volumes for the comb you are using.
Table 3-3. Sequencing Resuspension and Loading Volumes
Resuspension volume
Loading volume
2.
3-40
18
4 µL
4 µL
Comb Configuration (number of wells)
24
32/36
48
64
4–6 µL
4 µL
8–10 µL 8–12 µL
4–6 µL
4 µL
1.5–2 µL 1–1.5 µL
Vortex the samples and centrifuge briefly.
3 Using the Instrument in Sequencing Mode
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Applied Biosystems
3.
Heat samples at 90 °C for two minutes.
4.
Immediately place samples on ice until ready to load.
5.
Resuspend samples within 2 hours of loading.
Loading the Samples and Starting the Run
IMPORTANT
Be sure you load samples in the lanes specified in the Run window. The
tracker refers to the sample information to perform accurate lane
tracking. The sample information in the Run window when you start the
run is stored in the Gel file. Changes you make in the Run window after
you start the run are not implemented.
When you are loading many samples using a sharks-tooth comb and need to run
them in adjacent lanes, it is important that you load samples in alternate wells and
electrophorese the gel for 2 minutes, then rinse all the wells (step 1 on page 3-39)
and load the rest of the samples in the remaining unused wells. The automatic
lane tracker in the analysis software needs to have discrete spaces between the
samples to identify the lanes properly. Loading samples in adjacent lanes can blur
the definition between the lanes. Be sure to load strong (control) samples in the
outside (left-most and right-most) lanes to orient the tracker during analysis. For
best results, follow the procedure described below.
Note
In the following procedures, each button in the Run window has a
corresponding menu choice in the Instrument menu. You can either
click the button in the Run window or choose the command from the
Instrument menu.
For 48- and 64-well gels, make sure to deliver the samples to the bottom of the
wells. Use either a single- or multi-channel pipettor/syringe equipped with a flat
tip less than 0.3 mm in diameter (we recommend 0.17 to 0.20 mm). Do not
overload the lanes. Follow the recommendations in Table 3-3 for the allowable
loading volumes. If you use too much sample volume or quantity, you will
experience poor resolution.
March 2001
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Applied Biosystems
To load samples:
1.
If you are pre-running the gel, click Cancel in the Run window.
2.
Carefully flush all of the wells with running buffer.
Tip
3.
Place formamide or loading buffer in the well to the left of your first
sample lane, and in the well to the right of your last sample lane (the
comb creates wells to either side of the numbered wells). The
formamide in the buffer helps focus the bands in the first and last lanes.
Immediately load the odd-numbered samples into each of the
odd-numbered wells.
If you are loading fewer than half the samples allowed by the comb, skip
steps 6–10 and proceed to step 11. If you are loading a square-tooth comb,
you can load the wells consecutively and skip steps 6–10 and proceed to
step 11.
IMPORTANT
4.
Be sure to load strong (control) samples in the outside (left-most and
right-most) lanes. Avoid skipping more than one lane between samples.
It is better to leave extra empty lanes at the right end of the gel. The lane
tracker may not work properly if there are many empty lanes between
samples.
Click Run in the Run window to start the run.
A dialog box appears, allowing you to change the name and location of the
gel file created by the run.
5.
Click OK when you are satisfied with the name and location for the gel file.
Note
If a dialog box appears indicating you do not have enough room on your
hard disk to save the data generated by a run, delete files (particularly
Gel files) to create enough space, making sure to back them up on a
floppy disk or other storage device first, then click Run again.
6.
3-42
Electrophorese for two minutes so the entire sample enters the gel.
3 Using the Instrument in Sequencing Mode
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Applied Biosystems
7.
Click Pause in the Run window.
8.
Carefully flush all of the wells with running buffer to remove any residual
formamide from the previously loaded wells.
9.
Immediately load the even-numbered wells.
10. Click Resume in the Run window.
11. Choose Status from the Window menu to verify that electrophoresis and
scanning resume (refer to The Status Window on page 5-50).
If either electrophoresis or scanning does not resume, cancel the run and
restart it.
Figure 3-14 summarizes the buttons you click in the Run window for a typical run.
They are listed from top to bottom in the order you use them.
Note
March 2001
The tasks described in Figure 3-14 are detailed in the preceding pages.
3 Using the Instrument in Sequencing Mode
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Applied Biosystems
Button to click
Module in effect
Plate Check module
n/a
Pre-run module
Task to perform
Check plates and gel for
fluorescence
(Stops plate check scan)
Add buffer and check for leaks
Mount front heat-transfer
plate—Leon only
(Electrophorese gel without
data collection to equilibrate
temperature)
Pre-run module
Load first set of samples during
pause
Pre-run module
(Electrophorese samples into
gel without data collection)
Pre-run module
Load second set of samples
during pause
n/a
Run module
(Stops pre-run)
(Starts electrophoresis and data
collection)
Figure 3-14. Run window buttons used during a typical run
3-44
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Applied Biosystems
Observing Instrument Status and Data
After you begin electrophoresis and data collection, data appears on the
computer screen as it is collected. You can open up to four windows to view the
data and the instrument status. Only one window can be active at any given time,
but status windows still update when they are not active.
Note
If they are displayed, status windows continually update during a run.
They do so even when another application is running.
Open the data collection and status windows by choosing the name of the window
of interest from the Window menu.
Data Collection Status Windows
To observe instrument status, choose one of the following commands from the
Window menu. The page reference next to a window name indicates where you
can find detailed information about that window.
•
Status (refer to page 5-50)
•
Electrophoresis History (refer to page 5-57)
•
Log (refer to page 5-51)
Note
After the run is completed, you can choose Log from the Window menu
to display a comprehensive record of all error and status messages
generated during the run.
Real-time Data Windows
The data displayed in the real-time data windows (the Scan window and the Gel
window) represents light intensities collected by the data collection software from
the filter wheel and the PMT (photomultiplier tube) in the instrument. The
colors in the raw data windows represent the wavelengths of the dyes being
detected, rather than the bases being detected. This is explained in Filters and Color
of Data Display on page 5-52.
March 2001
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Applied Biosystems
To observe the data as it is being generated, choose one of the following from the
Window menu:
3-46
•
Scan (refer to page 5-55)
•
Gel (refer to page 5-56)
3 Using the Instrument in Sequencing Mode
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Applied Biosystems
Cleaning Up After the Run
After your run is complete, perform the following general cleanup steps:
•
Remove the gel from the instrument.
•
Clean the plates, comb, and spacers.
Removing the Gel from the Instrument
Classic
and
Leon
1.
Disconnect the electrode leads from both buffer chambers.
2.
Unlatch the beam-stop bar and pull it forward.
3.
Carefully remove the upper buffer chamber, glass plates, and heat plate
(Leon only) as one unit. Discard the buffer.
You can also set up a siphon system to drain the upper and lower buffer
chambers before removing them.
Stretch
4.
Remove the lower buffer chamber and discard the buffer.
5.
Clean up any liquid left in the electrophoresis chamber.
6.
Unclamp the plates from the upper buffer chamber.
7.
Rinse the buffer chambers with dH2O and allow them to air dry.
1.
Disconnect the electrode leads from both buffer chambers.
2.
Unlatch the beam-stop bar and pull it forward.
3.
Carefully remove the upper buffer chamber and glass plates as one unit.
Discard the buffer.
4.
Remove some of the buffer from the lower buffer chamber before
removing it.
This is especially important for 34-cm runs, as the buffer level is very high.
You can also set up a siphon system to drain the upper and lower buffer
chambers before removing them.
5.
March 2001
Remove the lower buffer chamber and discard any remaining buffer.
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Applied Biosystems
6.
Clean up any liquid left in the electrophoresis chamber.
7.
If you plan to change plate length next time, unplug the temperature
sensor, and then remove the heat plate, otherwise leave both in place.
8.
Unclamp the plates from the upper buffer chamber.
Note
9.
If the unnotched plate has been silated with a bind-silane compound,
use 0.1 N HCl or NaOH to remove the gel from the silated area, or
scrape it off gently.
Rinse the buffer chambers with dH2O and allow them to air dry.
Cleaning the Plates, Comb, and Spacers
Caution
3-48
Avoid contacting the glass plates with metal. Scratches caused by
metal objects, such as drying racks or spatulas, weaken the glass
and reduce its usable life.
1.
Gently push a plastic wedge almost all the way in at the bottom of the plates
(avoid the top notches) and pry the plates apart. Do not use too much
force or the plates might chip. Remove the sharks-tooth comb and gel
spacers.
2.
Lay a large Kimwipe on the gel and roll it up, lifting the gel off the plate,
and discard appropriately.
3.
Rinse the plates with water. Rub with a gloved hand to dislodge remaining
gel bits, then rinse again with water.
4.
Wash with a detergent such as Alconox that will not leave a residue, rinse
with dH2O, and allow to dry.
5.
Clean the comb and spacers and store them.
3 Using the Instrument in Sequencing Mode
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Applied Biosystems
Note
6.
March 2001
Do not expose combs or spacers to moisture any longer than is
necessary to clean them. Store combs and spacers carefully to avoid
adding blemishes. Even very small bumps or bends can ruin the combs
and spacers.
Wipe any spilled buffer off the heat plate.
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Applied Biosystems
Analyzing the Data
If you choose Auto Analyze for your samples in the Sequence Run window, the
data collected on the ABI PRISM 373XL is automatically analyzed by the
Sequencing Analysis program. For complete instructions on using the analysis
program, refer to the ABI PRISM DNA Sequencing Analysis Software User’s Manual that
came with the software.
Automatic data analysis occurs approximately 6.5 to 16 hours after you start data
collection (the amount of time depends on the module you chose). The analysis
software prints electropherograms (if you selected AutoPrint in the Sample
Sheet) and displays the gel image on the computer screen.
Color of Data Display and Output
The colors in the raw data windows represent the wavelengths of the dyes being
detected, rather than the bases being detected (see Filters and Color of Data Display
on page 5-52).
The analysis program converts the information collected by the data collection
program so that after analysis the colors are the same for each base. The colors on
all displays of analyzed data, including printed electropherograms, are as follows:
C = Blue
Note
3-50
A = Green
G = Yellow
T = Red
When printed and when shown against white on the screen, G is black,
so it is easier to see.
3 Using the Instrument in Sequencing Mode
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Applied Biosystems
Reading an Electropherogram
March 2001
•
You can view the electropherograms in the analysis program as Raw or
Analyzed data (data is also printed in these formats). Raw and analyzed
data in a Sample file correspond to data/scan points collected.
•
Information from the Sample Sheet appears at the top of printed
electropherograms.
•
The colors of the peaks and the letters above the peaks correspond to the
appropriate bases.
•
An N above a peak means that the software could not confirm the identity
of that base.
•
In the analyzed data, the numbers above the colored peaks indicate
analyzed base positions, counted from the first analyzed data point.
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Archiving the Data
For best performance, keep as few files as possible on your hard disk. Do not
archive gel files and run folders on your hard disk. It is important that you copy
all files you want to save onto a backup medium before beginning another
sequencing run, both to preserve the files and to free disk space.
Files Created During a Run
When you set up a run, the data collection software creates a Run folder and a Run
file. During the run, the program automatically creates and saves two files: the Gel
file and the Run Log file. After the run, the analysis program creates Sample and
sequence (.Seq) files.
Run Files
Each Run file stores information associating specific samples with specific lane
positions. It also stores information about the Sample Sheet and module
associated with the run. Run files are small enough to store on a disk.
Gel Files
Gel files contain the raw data acquired by the data collection program and
typically take up 10–55MB of disk space. A separate Gel file is created in the Run
folder each time you perform a run. Because Gel files take a substantial amount
of space, you should delete them from your hard disk after you have obtained
satisfactory Sample files. You do not usually need to keep gel files once the lane
tracking is verified and sample files are created.
Use magnetic tapes, removable cartridge drives, or optical drives to archive gel
files. Gel files are too large to fit on floppy disks.
IMPORTANT
Do not discard any gel file until you have verified the tracking and taken
corrective action if necessary.
Run Log Files
Run logs provide a good record of your runs. Individually, Run Log files are small
enough to archive on floppy disks.
3-52
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Applied Biosystems
Sample files
Use disks, a magnetic tape drive, a removable cartridge drive, or an optical drive
to archive sample files. A 1.4 MB, high-density disk holds about six files. A Sample
file is 150–200KB in size, depending on the length of the sequencing run;
however, Sample files for 388 runs are twice as large. Save a Sample file when you
feel confident that the channel selections (tracking) are correct for the sample.
.Seq files
.Seq files show the base letter sequence only (or might contain a header,
depending on the format you choose). You can open .Seq files from word
processing programs and print them. The Preferences dialog box in the
Sequencing analysis program allows you to choose the file format before analysis.
Other Files
Other files provide information for a run that you might have customized and
should therefore save. These include the Sample Sheet, analysis settings, or matrix
files you create.
Sample Sheet Files
You can reuse the Sample Sheet, and it is a good reference after data collection.
Individually, Sample Sheet files are small enough to archive on disks.
Custom Analysis Settings and Instrument Files
If you have created customized files, you should keep backup copies of the files on
a separate medium. Each of these types of files alone is small enough to store on
a disk.
March 2001
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300 I'M INVISIBLEP
Applied Biosystems
4 Using the Instrument in GeneScan Mode
Contents
Introduction
Before You Set Up the Instrument
Preparing Samples
Experimental Design Considerations
Working with Overlapping Allele Size Ranges
Multiplexing the Polymerase Chain Reaction
Optimizing Salt Concentrations
Multiplexing Samples in a Single Lane
Sizing and Quantifying Sample Fragments
Setting Up the Software
Setting Up the Instrument
Preparing the Gel for Loading
Reagents and Supplies Needed For Preparing the Glass Plates
Preparing the Electrophoresis Chamber
Model-Specific Notation
Setting Up the Software
Setting up the Analysis Software
Starting the Data Collection Software
Creating User-Specific Module Files
Setting Default Parameters (Preferences)
Creating the GeneScan Sample Sheet
Creating a Run File
Checking the Plates
Cleaning the Plates or Skipping Lanes
Skipping Lanes
Checking the PMT Gain Setting
Verify Run Mode
First Time Run
Inserting the Sharks-tooth Comb
Completing Instrument Setup
Preparing and Loading Samples
March 2001
4 Using the Instrument in GeneScan Mode
4
5
5
5
6
6
6
7
7
8
9
9
9
11
11
19
19
20
20
22
24
26
30
31
32
32
32
32
34
36
41
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Applied Biosystems
Pre-running the Gel
Preparing Samples for Electrophoresis
Loading the Samples and Starting the Run
Observing Instrument Status and Data
Data Collection Status Windows
Real-time Data Windows
Cleaning Up After the Run
Removing the Gel from the Instrument
Cleaning the Plates, Comb, and Spacers
Analyzing the Data
Evaluation Checklist
Characteristics of Good Data
Characteristics of Poor Data
No Peaks
Too Many Peaks— Noisy Data
Smeared or Fuzzy Bands
Troubleshooting Table
Archiving the Data
Files Created During a Run
Run Files
Gel Files
Run Log Files
Sample Files
Other Files
Sample Sheet Files
Custom Analysis Settings and Matrix Files
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Prepare and label samples
Pour gel
Polymerize
~2 hr
Restart Macintosh
Set up software
Load gel on instrument
Run plate check
Cancel plate check
Add buffer,
Start pre-run
Pre-run
~5 min
Run samples
into gel
~ 2 min
Mix sample with loading buffer size std
Heat sample
Cancel Pre-run
Load samples
Start Run
Run
~ 3–8 hr
Observe status
Analyze and print (automatic)
Clean up
Figure 4-1. Flow chart for electrophoresing/collecting data on ABI PRISM 373 DNA
Sequencer with XL Upgrade
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Applied Biosystems
Introduction
This section guides you through the process of electrophoresing your DNA
samples and collecting the resulting GeneScan data on the ABI PRISM 373XL
DNA Sequencer. Figure 4-1 shows the recommended flow of the process.
Although this section is detailed, it does not provide complete details for each
procedure. Use it as a guide and refer, as necessary, to the cross-referenced pages
for more detail.
Note
If you have previous experience using the Model 373, you should read
through these steps. Data collection software has been streamlined.
You no longer need to enter any commands on the instrument itself.
The information in this section applies to GeneScan applications. Instrument
setup consists of similar steps for both Sequencing and GeneScan applications.
The main differences are in completing the Sample Sheet and the Run file.
For further information about the analysis performed for each application, refer
to the GeneScan Analysis software manual.
To read details about the data collection software and the instrument hardware
refer to Sections 5, 6, and 7.
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Applied Biosystems
Before You Set Up the Instrument
Preparing Samples
Make sure you have prepared your samples as described in the GeneScan 672 User’s
Manual, or in the appropriate kit protocol. Before you set up your instrument,
perform the following procedures:
•
Purify and quantitate DNA samples.
•
Label DNA fragments with fluorescent dyes.
•
Prepare the appropriate gel.
Experimental Design Considerations
Fluorescent labeling enables you to analyze many independent fragments in the
same lane using either color or fragment length to distinguish between fragments.
Some factors to consider when you design your experiments are:
March 2001
•
Working with overlapping allele size ranges
•
Multiplexing the Polymerase Chain Reaction (PCR)
•
Optimizing salt concentrations
•
Sizing and quantifying sample fragments
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Applied Biosystems
Working with Overlapping Allele Size Ranges
If the expected size ranges of PCR products from different primer pairs overlap,
you can do one of the following to differentiate between them:
•
Label primer pairs with different dye colors
•
Choose new primer sites to alter PCR product fragment lengths
•
Load overlapping products in different gel lanes
For more information on labeling techniques, see the GeneScan 672 Software User’s
Manual.
Multiplexing the Polymerase Chain Reaction
You can multiplex the Polymerase Chain Reaction (PCR) by adding more than
one pair of primers into the same reaction tube, and then performing the PCR. If
you multiplex, you should first check primers for compatibility considerations,
such as excessive regions of complementarity, or similar melting temperatures.
Once you have chosen compatible primer pairs, you should then test the pairs for
successful co-amplification. Multiplexing often requires that you optimize
reaction conditions, and sometimes requires that you redesign primers. For more
information, refer to Appendix E, Optimizing PCR.
Optimizing Salt Concentrations
Multiplexing by co-loading several PCR products in a single lane improves
throughput, but can also increase the salt concentration in your samples. High salt
concentration in your samples can inhibit complete denaturation of
double-stranded DNA. As a result, the ABI PRISM 373 DNA Sequencer with XL
Upgrade detects less DNA, weakening the signal. To prevent high salt
concentrations from negatively impacting your run, make sure you use at least
equal volumes of formamide and PCR solution in each of your samples.
To increase the signal intensity, do one of the following:
•
Combine the samples, precipitate the product, and resuspend the samples
•
Use a spin column
•
Add 1 or 2 extra PCR cycles in your protocol to produce more product per
µL of PCR reaction
Merely evaporating your samples increases the concentration of salt, which makes
it more difficult to denature the DNA in subsequent steps.
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Applied Biosystems
Multiplexing Samples in a Single Lane
In addition to multiplexing the PCR, you can multiplex samples on the ABI PRISM
373 DNA Sequencer with XL Upgrade by performing multiple PCRs in separate
reaction tubes, pooling the PCR products together, and co-loading multiple
samples in a single lane of the gel.
If you multiplex samples, consider the following when designing your
experiment:
•
Use different colored ABI PRISM dyes for labeling each sample you co-load
in a single lane, if they overlap in size.
•
Because each dye has a different absorption maximum, the intensity of
fluorescent signal emitted differs for each dye:
Filter Set A
ROX (lowest signal intensity)
TAMRA
JOE
5-FAM (highest signal intensity)
Filter Set B
TAMRA (lowest signal intensity)
HEX
TET
6-FAM (highest signal intensity)
When labeling fragments, use a greater concentration of dye for dyes of lower
intensity. For example, you would need to use approximately eight times as much
ROX dye as 6-FAM or TET dye to generate signals of equal intensity. Also, when
choosing combinations of dyes for sample labeling, choose dyes that can be
detected by the same filter set: A or B.
Sizing and Quantifying Sample Fragments
When preparing samples, consider how choice of dye labels and size standard
affects analysis of fragment size and quantity. For the same study, use the same size
standard and dye label for all samples.
When analyzing your results, compare only peak heights, peak sizes, or peak areas
of fragments labeled with the same dye label. For example, compare HEX-labeled
fragments only to other HEX-labeled fragments.
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Applied Biosystems
Setting Up the Software
While the gel is polymerizing, you can set up the Sample Sheet and the Run file
for the data collection software. Refer to Setting Up the Software on page 4-19.
If you are running GeneScan applications or wish to use custom analysis settings,
you should also set up the conditions that control data analysis before you start a
run. To do so, refer to the user’s manual provided with your GeneScan analysis
software.
IMPORTANT
If you choose automatic analysis and want the GeneScan Analysis
software to use settings other than the default, customize the analysis
settings before you start data collection.
If you specify automatic analysis in the data collection program, the GeneScan
Analysis program starts automatically at the end of the run to process the collected
data.
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Applied Biosystems
Setting Up the Instrument
Pour your gel as described in Section 2, and allow it to polymerize for two hours
before proceeding with this section.
IMPORTANT
Make sure to use the appropriate gel concentration for the gel length
and run type.
Preparing the Gel for Loading
Note
Wear clean laboratory gloves throughout this procedure, both for your
protection and to avoid transferring fluorescent contaminants from your
hands to the glass plates.
Reagents and Supplies Needed For Preparing the Glass Plates
March 2001
•
A single-edged razor blade or a second casting comb
•
1.5-L, 1X Tris Borate EDTA buffer (2 L for 34-cm gels), pH 8.3 (refer to
10X TBE Stock Solution on page 2-10)
•
A clean, 10–50 mL syringe and a 22 G needle
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To clean the plates:
1.
Remove the clamps on the gel casting (single-well) comb or square-tooth
comb, and the tape from the edges of the plates.
2.
Rinse the glass plates with cold water.
a.
Rinse rinse both sides and edges of the glass plates thoroughly with
cold tap water to remove dried acrylamide and urea.
b.
Rinse the well and plates with dH20 and allow the plates to air dry.
To remove the square-tooth comb:
1.
Before you remove the square-tooth comb (Figure 4-2), lay the appropriate
plastic overlay strip on the clean glass. Align the well outlines on the overlay
strip with the white teeth of the comb.
Figure 4-2. Square-tooth comb
2.
Carefully lift the edge of the square-tooth comb.
To avoid suction, pry gently with a razor blade or plastic wedge, or squirt
deionized water over and around the comb.
3.
Using both hands, apply gentle, even pressure and slowly pull out the
comb.
4.
Clean the comb area with deionized water and Kimwipes.
Note
4-10
If you are using the Sharks-tooth comb: refer to To prepare the plates
for loading on the instrument: on page 3-7 for removing the casting
comb; refer to Inserting the Sharks-tooth Comb on page 3-34.
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Applied Biosystems
Preparing the Electrophoresis Chamber
WARNING
ELECTRICAL SHOCK HAZARD. The Model 373 contains a high
voltage power supply. This instrument has been designed with
safety features in the door to disconnect the power supply when
the door is open. Although it is designed for safe operation, please
follow procedures as prescribed. As with any electrophoresis
apparatus, care should be taken during instrument operation and
when handling electrodes and liquids.
WARNING
ELECTRICAL SHOCK HAZARD. Wait at least 5 seconds after
opening the door before touching the electrodes in the
electrophoresis chamber. Capacitors hold an electrical charge
even after the power supply is disconnected.
Model-Specific Notation
This manual applies to three configurations of Model 373 DNA Sequencers:
Classic, Leon, and Stretch.
•
Classic. This configuration includes 370s with Macintosh upgrade and the
373A. Classic models have electrophoresis chambers which support 24-cm
WTR distances.
•
Leon. This configuration has an electrophoresis chamber which supports
6-, 12-, and 24-cm WTR distances.
•
Stretch. This configuration supports 6-, 12-, 24-, 34-, and 48-cm WTR
distances and is easily identified by its extended door.
Note
All three instrument configurations must have the five-filter wheel
capability.
Most of the information in this manual applies to all three configurations,
however, some sections of the manual apply to only one or two of these
configurations. Those sections are preceded by the name of the configuration
(Classic, Leon, or Stretch) to which they apply.
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To prepare the electrophoresis chamber:
Classic
1.
Open the door to the Model 373 and make sure the heating plate is dry.
2.
Place the lower buffer chamber in the bottom of the electrophoresis
chamber with the electrode to the front. The upper chamber will not be
needed until later.
Upper buffer chamber
Glass plates
Model 373A,
side view
Beam-stop bar
Lower buffer
chamber
Figure 4-3. Placement of the Buffer Chambers, glass plates, and beam-stop
bar in the electrophoresis chamber of the Model 373 (Classic)
3.
Pull the black beam-stop bar in the electrophoresis chamber toward you
until it stops (Figure 4-3). Rest the bottom edge of the plates on the
notched supports in the bottom of the lower buffer chamber, with the
notched plate facing away from you. Stand the plates in a vertical position.
Press them firmly against the heating plate, and then push the beam-stop
bar up against the plates. Center the plates in relation to the beam-stop bar,
and then lock the beam stop bar in place by sliding the latches down along
the outer edges.
IMPORTANT
4-12
It is very important to center the plates. Centering the plates ensures
that each sample migrates within the read region of the laser.
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Applied Biosystems
Note
Leon
1.
The beam-stop bar is removable. In normal operation you should not
need to remove it. To install the beam-stop bar if it is removed, line up
the alignment pins in the alignment grooves and slide the beam-stop
bar into place.
Open the door to the Model 373.
Upper buffer chamber
support
24 cm
12 cm
6 cm
Beam-stop bar
Beam-stop
swivel clamps
Glass plate support
Lower buffer chamber
Figure 4-4. Model 373 (Leon) Electrophoresis Chamber
March 2001
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Applied Biosystems
4-14
2.
Place the lower buffer chamber in the bottom of the electrophoresis
chamber with the electrode to the front. The upper chamber will not be
needed until later.
3.
Adjust the upper buffer chamber supports to accommodate the different
plate lengths (6, 12, and 24 cm) (refer to Figure 4-4 on page 4-13. Loosen
the screws and slide the brackets to the correct position as indicated by the
well-to-read length printed on the supports, and then tighten the screws.
4.
Place the glass plates into the instrument as follows:
a.
Pull the black beam-stop bar in the electrophoresis chamber towards
you until it stops (Figure 4-4 on page 4-13).
b.
Rest the bottom edge of the plates on the notched supports in the
bottom of the lower buffer chamber, with the notched plate facing
away from you.
c.
Stand the plates up in the vertical position. Press them firmly against
the back of the chamber (avoid touching the laser read region, and
then push the beam-stop bar up against the plates.
d.
Center the plates with relation to the beam-stop bar and then latch
the bar in place.
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Applied Biosystems
Stretch
1.
Open the door to the Model 373. The electrophoresis chamber looks like
the one shown in Figure 4-5.
34
-cm
Upper buffer chamber
supports and shelf
WT
R
24
-cm
WT
R
12
-cm
WT
R
Beam-stop bar
R
m
6-c
WT
Gel plate rests
(ABS blocks)
Lower buffer chamber
Figure 4-5. Model 373 (Stretch) Electrophoresis Chamber
2.
If you are running a 24- or 34-cm gel, place the white plastic upper buffer
chamber shelf on the appropriate support on the inside of the
electrophoresis chamber (see Figure 4-5).
If you are running a 12-cm gel, place two white plastic upper buffer
chamber shelves on the support.
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Applied Biosystems
If you are running a 6-cm gel, then no shelves are required.
3.
For 34- and 24-cm gels, slide the heat plate into place, as follows:
a.
Choose the correct heat plate for the glass plates you are using.
b.
Make sure the electrical wires for the temperature sensor are at the
top, with the plate clamps toward the front. Align the grooves on
either side of the heat plate with the heat plate guides on the
chamber.
c.
Slide the heat plate down until it rests on the bottom of the chamber.
d.
Plug the temperature sensor on the heat plate into the socket at the
top left of the chamber.
e.
Plug the heat plate ground into the socket at the top right of the
chamber.
For 6 and 12 cm WTR gels, use the temperature sensor alignment brace.
IMPORTANT
4.
Make sure the heat plate ground is connected before continuing.
Adjust the gel plate rests in the lower buffer chamber, if necessary (see
Figure 4-6). The gel plates rests are located in the bottom of the buffer
chamber and serve to elevate the glass plates. In order for the 34 cm plates
to rest in the correct position, they must sit higher in the buffer chamber.
All other plates rest in the lower position.
Each gel plate rest consists of several parts, a large ABS block on the
bottom, a shorter ABS block on top of it, and an adjustable thumb screw.
When the thumb screw is loose, the small block can be moved forward and
back, so that the plates rest either on one block, or on two blocks (see
Figure 4-6).
4-16
a.
Loosen the thumb screws.
b.
Slide the blocks toward the back of the chamber if you are using
34 cm plates, so the plates rest in the high position. Slide the blocks
toward the front of the chamber for all other gel lengths so the plates
rest in the low position.
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Applied Biosystems
c.
Tighten the thumb screws.
Thumb Screw
a. For all run lengths except 34 cm. With
the movable blocks toward the front of the
electrophoresis chamber, the glass plates
rest lower in the buffer chamber.
Front of electrophoresis chamber
Glass Plates rest here
Thumb Screw
b. For 34 cm runs only. With the movable
blocks toward the back of the
electrophoresis chamber, the glass plates
rest higher in the buffer chamber.
Front of electrophoresis chamber
Figure 4-6. Adjustable gel plate rests
March 2001
5.
Place the lower buffer chamber in the bottom of the electrophoresis
chamber with the electrode to the front. The upper chamber will not be
needed until later.
6.
Unfasten the latches on either side of the beam-stop bar and swing the
beam-stop bar forward, toward you.
7.
Place the glass plates in the electrophoresis chamber in front of the heat
plate and read window, with the bottom of the plates resting on the gel
plate rests.
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Applied Biosystems
a.
Center the glass plates carefully, or the beam-stop bar will not fasten.
b.
Press the glass plates against the heat plate and close the beam-stop
bar.
c.
Fasten the latches at either side of the beam-stop bar.
IMPORTANT
4-18
Do not touch the gel plates within the read region.
4 Using the Instrument in GeneScan Mode
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Applied Biosystems
Setting Up the Software
Before starting electrophoresis, you must ensure there is sufficient space on your
hard disk and restart the Macintosh. If you delete files, you must restart the
Macintosh. You must also set up certain files in the data collection software:
•
Set preferences
•
Record sample information in the GeneScan Sample Sheet
•
Create a GeneScan Run file
Note
These tasks are best performed while you are waiting for the gel to
polymerize.
You can simplify creation of the files by setting preferences for certain parameters
in each file.
The procedures described here do not include detailed descriptions of the data
collection software. They are intended to give you a guide for steps you perform
and the order in which to perform them. For specific information about the
software and how to complete specific fields, refer to Section 5.
You may not need to change these default parameters for future runs, if you always
run the same or similar parameters. However, it is important to verify these
defaults, since they determine the names and locations of your data.
If you desire more detail, the steps described here refer you to the specific pages
in Section 5 where you can find related information.
Setting up the Analysis Software
If you are running GeneScan applications or wish to use custom analysis settings,
you should set up the conditions that control data analysis before you start a run.
To do so, refer to the User’s Manual provided with your GeneScan analysis
software.
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Applied Biosystems
Starting the Data Collection Software
1.
Choose Restart from the Special menu in the Finder to restart your
Macintosh.
2.
The data collection software should automatically open upon restarting
the Macintosh. If it does not open, double-click the icon to open the
program. Refer to To make the data collection program open at startup: on page
5-13 for instructions to open the collection program at startup.
Note
When you start the ABI PRISM 373 DNA Sequencer with XL Upgrade
data collection program, the program searches for the current version
of firmware on the Translation Interface Processor (TIP). If none is
found, the program automatically copies the firmware image file (stored
on the Macintosh) to the TIP. If the TIP’s version of firmware is different
from the collection software version, a dialog box appears, allowing you
to continue or send a new firmware to the TIP. Click Send New
Firmware to update your firmware version.
Creating User-Specific Module Files
The ABI PRISM 373 DNA Sequencer with XL Upgrade DNA Sequencer upgrade is
shipped with module file templates for plate check, prerun, sequencing and
GeneScan runs. Table 4-1 shows the default parameters for the modules file
templates.
IMPORTANT
The parameters in Table 4-1 apply only to 24-cm WTR Classic or Leon
denaturing 6% acrylamide gels. You must make appropriate modules
for all other, i.e., 6-, 12-, and 34-cm plate lengths and for native
applications.
Table 4-1. Module File Default Parameters
Module
Plate Check
Pre Run
Seq Run
4-20
Field
(Volts)
-2500
2500
Power
(Watts)
-30
30
Current
(mAmps)
-40
40
Laser Power
(mWatts)
40
40
40
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Run Time
(Hours)
0.50
0.33
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Module
GS Run
Field
(Volts)
2500
Power
(Watts)
30
Current
(mAmps)
40
Laser Power
(mWatts)
40
Run Time
(Hours)
12
You must create user-specific module files the first time the instrument is used and
for each different run condition. Once you modify a module file template, you
can save it and use it each time a similar run is performed. Refer to Table 1-3 on
page 1-15 and Appendix B for suggested module file and Run parameters.
To modify a module file:
1.
Choose New from the File menu.
2.
Click the Sequence Run or GeneScan icon in the window that appears.
3.
Choose the module file you wish to modify from the Run Module pop-up
menu.
Note
You do not need to complete the other information in the Run window
to modify a module file.
4.
Click the small document icon next to the Run module pop-up menu.
Figure 4-7. Module settings dialog box
March 2001
5.
Type new number in the applicable entry fields. Refer to Table 1-2 on page
1-14 for recommended run parameters.
6.
Save the changes.
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• Save as Default: To save the changed parameters as defaults for the
module without changing the module name, click Save as Default. The
new settings are permanently saved and the name of the module file
remains unchanged.
• Save Copy in…: To save the changed parameters as defaults, click Save
Copy in. Type in a new name for the module file and save the file in the
Modules Folder. The new settings are permanently saved and the name
of the module file is changed.
Note
You will not be able to select your new module file in the pop-up menu
until you save and close the Run window, then reopen the saved Run
file or create a new Run file.
• Save: To use the changes as a one-time override for the current run,
click Save.
Note
Custom module files are stored on your hard disk. and can be used for
any subsequent run. If you reload your collection software, however,
you must re-create your custom modules.
Setting Default Parameters (Preferences)
You might only need to set default parameters prior to the first run. If you are
satisfied with the defaults stored in the instrument, skip to page 4-24 to create a
Sample Sheet and a Run file. However, if more than one person uses the same
instrument, we recommend you set or verify all preferences prior to starting a run.
You can set several types of preferred defaults:
4-22
•
Folder locations: The ABI PRISM 373XL data collection software uses
specified folders for data storage. This option allows you to change the
folders it uses. For information about changing folder locations, refer to To
change one of the data storage folders: on page 5-17.
•
Default File names: The data collection program automatically names new
files and folders created during a run. You can specify the prefix and suffix
of each type of file (see Setting File Name Preferences on page 5-18).
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Applied Biosystems
•
GeneScan Sample Sheet defaults: You can set defaults for certain parameters
on the Sample Sheet. Since the Sample Sheets differ for Sequence Analysis
and GeneScan (fragment) Analysis, the parameters for which you can set
defaults also differ somewhat. To set defaults for the GeneScan Sample
Sheet, refer to To set Sample Sheet defaults for GeneScan analysis applications: on
page 5-25.
•
GeneScan Run defaults: You can also set defaults for certain parameters in
the Run file. As with the Sample Sheet defaults, separate defaults apply for
sequence runs and GeneScan analysis runs. To set defaults for sequence
Run files, refer to Setting Defaults for GeneScan Analysis Applications on page
5-25.
•
General settings: The ABI PRISM 373XL instrument is normally attached to
the Modem port of the Macintosh computer. If you need to change the
connection, you can specify the change using this preference choice. Also
use this choice to reset the global serial number, which is a number used
by the file naming defaults (refer to General Settings on page 5-27). To
change general settings, refer to To change the general settings: on page 5-28.
•
Dye indicators: Each of the four dyes used during a run has a preset code and
color. You can change the color assigned to each dye, and change the color
that appears in the plot when it is printed. To do so, refer to To set dye
indicator preferences for the analysis applications: on page 5-29.
To set preferences:
1.
March 2001
Choose Preferences from the Window menu and choose the appropriate
option from the submenu that appears.
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Applied Biosystems
Figure 4-8. Preferences submenu
2.
Enter or change the defaults, as described on the pages noted above.
3.
Click OK to accept the changes.
Creating the GeneScan Sample Sheet
Before starting a run you must record sample information in the GeneScan
Sample Sheet and the Run file. Once you have set up the Sample Sheet, you can
automatically copy sample information into the Run file, which associates sample
information (name and type of analysis) with each lane position in the gel.
Separate Sample Sheets exist for sequencing and GeneScan analysis applications
(see Figure 4-9). Be sure to choose the correct type for your application.
If a Sample Sheet containing the correct information already exists, you can use
it. In most cases, you need to create a new Sample Sheet for each run. You can
enter the information directly or export information from a database in
tab-delimited format and import it into the Sample Sheet.
To create a GeneScan Sample sheet:
1.
Choose New (z-N) from the File menu.
2.
Click the GeneScan Sample Sheet icon in the window that appears.
The Sample Sheet is similar to a spreadsheet or ledger. It accepts more text
than fits in the visible fields and automatically scrolls as you type your entry.
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Applied Biosystems
To view the beginning of a long entry, use the keyboard arrow keys. Many
of the cells have default values that are automatically filled in for each
sample.
3.
Enter the necessary information. For details about completing the Sample
Sheets (including information about importing from a database), refer to
Creating and Editing a Sample Sheet on page 5-32.
IMPORTANT
Although the Sample Sheet has 72 lanes available, fill in the sample
information only for the number of lanes you are using.
IMPORTANT
Do not mix sequence analysis and GeneScan analysis samples on the
same Sample Sheet or the same run.
4.
When you are finished, choose Save from the File menu.
The dialog box that appears shows the default file name and storage
location as defined in the File Names and Folders locations preferences,
respectively. You can change the file name if you wish.
5.
Click Save.
Make sure you are saving the file into the Sample Sheet folder, so the name
will appear in the pop-up menu.
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Applied Biosystems
6.
Choose Close from the File menu, or click the close box.
Specify sample names
for each color
Specify size
standard color
Enter sample information and
additional comments
Figure 4-9. Blank GeneScan Sample Sheet
Creating a Run File
In the Run file you specify the lanes in which the samples will run and parameters
to be used for both the pre-run and the run. If a Run file exists that contains the
proper parameters, you can copy it to a new folder and use it. In most cases you
need to create a Run file for each new run.
Separate Run windows exist for sequencing and GeneScan analysis applications.
They look almost the same; the GeneScan analysis version has an additional
(Analysis Settings) column. Be sure to choose the correct type for your
application.
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Applied Biosystems
To create a Run file:
1.
Choose New from the File menu.
2.
Click the GeneScan Run icon in the window that appears.
Small document icons open the selected
*module or Sample Sheet for review
Specify length of run
Run control
buttons
Pop-up menus in
these panels allow
you to choose the
modules
(parameters), the
associated
Sample Sheet, the
number of wells,
the separation
distance (WTR),
and the matrix for
the run
Lane numbers cannot be edited
Figure 4-10. GeneScan Run window
3.
Choose the parameters for the run from the pop-up menus.
Note
Any changes you make in your preferences setting will not take effect
until you open a new Run window.
a.
March 2001
Choose modules to be used for the plate check, the pre-run and the
run in the Module pop-up menus.
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b.
Choose the Sample Sheet from the Sample Sheet pop-up menu.
When you choose the Sample Sheet, the information about the lanes
in which the samples are to run fills in automatically.
c.
Choose the number of lanes and the separation distance from the
Lanes pop-up menu and the WTR pop-up menu, respectively.
d.
Choose XL Scan or Full Scan in the Run Mode pop-up menu. Use the
table below to determine the appropriate run mode for your lane
configuration.
Lanes
24
24
32
34
36
36
48
50
64
66
Note
Run Mode
Full Scan
Full Scan
Full Scan
Full Scan
Full Scan
Full Scan
XL Scan
XL Scan
XL Scan
XL Scan
If you select a run mode that is incompatible with the selected lane
configuration, the lane popup menu displays <none>.
e.
4.
Comb
Sharks-tooth
Square-tooth
Sharks-tooth
Square-tooth
Sharks-tooth
Square-tooth
Sharks-tooth
Square-tooth
Sharks-tooth
Square-tooth
Choose a matrix file from the Gel Matrix pop-up menu.
Enter the length of the run in the Collect Time entry field.
Refer to Application Protocols in Appendix C of the 672 User’s Manual for
collection times.
5.
Enter your name in the Operator entry field.
The sample information for each lane is entered automatically in the grid
at the bottom of the Run window when you choose a Sample Sheet.
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Applied Biosystems
Note
6.
You cannot edit the Sample Sheet information in the Run window.
Make any changes in the Sample Sheet, save it, select <none>, and
then select the Sample Sheet again in the Run window to make the
changes effective.
Choose the appropriate filter set: A or B.
IMPORTANT
7.
Failure to select the appropriate filter set results in the loss of data.
Select the Auto Analyze and Auto Print checkboxes if you want the analysis
program to analyze and send data to the printer.
When you finish completing the information in the Run file, do not close the Run
file window. You can use it to perform a plate check, as described next.
The options available to you at this time are:
•
Save the file for future use, then click the close box to close it.
•
Start a plate check by clicking the Plate Check button.
To save the file, choose Save from the File menu. To perform a plate check, see
page 4-30.
For more information about creating the Run file, refer to page 5-40.
March 2001
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Applied Biosystems
Checking the Plates
Scan the gel and plates before adding buffer and loading samples, to ensure that
no peaks are produced by fluorescent particles on the glass plates or in the gel.
Click Plate Check after you choose the Plate
Check module
Figure 4-11. Completed GeneScan Run window
To check the plates:
Note
1.
Note
4-30
For 6-cm and 12-cm runs on Stretch-configured instruments, the
temperature sensor alignment brace must be connected before runing
the plate check.
Click Plate Check in the Run window.
If a dialog box appears indicating you do not have enough room on your
hard disk to save the files created by a run, delete files (particularly Gel
files) to create enough space, making sure to back them up on a disk
or other storage device first, then click Plate Check again.
4 Using the Instrument in GeneScan Mode
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Applied Biosystems
The laser scans the plates without electrophoresis to detect any unwanted
fluorescent material in the read region.
2.
Observe the Scan window that appears.
The Scan window should show a relatively flat line across the screen in each
of the four colors. Typically, the lowest line is blue, followed by the green,
yellow (black), and red lines. If the scan line is flat, then the plates are
clean. Proceed to Checking the PMT Gain Setting on page 4-32.
If you see peaks in the Scan window, clean the glass plates again, as
described below.
WARNING
LASER HAZARD. Do not look directly into the laser beam or allow
a reflection of the beam to enter your eyes. Exposure to direct or
reflected laser light at 40 mW for 0.1 seconds can burn the retina
of the eye and leave a permanent blind spot.
Cleaning the Plates or Skipping Lanes
Peaks in the scan lines could indicate dirt on the glass or contaminating
fluorescence in the gel. To eliminate dirt on the glass, clean the plates and scan
them again. If peaks still appear and you wish to use the gel, avoid loading samples
in the lanes where the peaks appear.
To clean the plates and re-scan:
March 2001
1.
Click Pause to pause the plate check.
2.
Open the door of the instrument and unlatch the beam stop bar and
remove the plates.
3.
Carefully reclean both sides of the glass in the laser read region with dH2O
and a lint-free tissue or ultra-pure pressurized air.
4.
Replace the plates, center them, and latch the beam stop bar in the
electrophoresis chamber.
5.
Click Resume and check the plates again.
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If the peaks do not disappear after cleaning the glass, the gel mixture or buffer
might contain particles. If you wish to use the gel, avoid loading samples in the
lanes where the peaks appear.
Note
Occasionally, peaks in the scan window are caused by a dirty beam
stop bar, rather than by dust or particulates in the gel. Clean the beam
stop bar with dH2O and (optionally) a thin layer of oil, if the peaks do not
disappear after cleaning the glass.
Skipping Lanes
If a plate check shows peaks in some lanes of a gel even after cleaning, the Scan
window indicates which channels have contaminating fluorescence in them. Refer
to Table 3-2 on page 3-29 as a guide for estimating the channel number/lane
assignment correlations. This table should be used only as an approximation, as it
determines the center of each lane for sharks-tooth comb configurations and may
differ slightly for square-tooth combs.
If the contaminant peaks are close to the edge of a lane, you may want to skip two
lanes to ensure that there is no contamination in the lanes you load.
Checking the PMT Gain Setting
Verify Run Mode
The PMT gain setting differs for each of the run modes (XL Scan and Full Scan).
Verify that you have selected the correct run mode in the Run window before
checking the PMT gain setting.
First Time Run
The PMT gain settings are stored in a Calibration file. If this is the first time the
instrument is being used, you must make and send a Calibration file. Refer to
Section 5 for instructions on how to make and send a Calibration file.
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Applied Biosystems
Recommended PMT Gain
The photomultiplier (PMT) gain should be set such that the lowest scan line
(usually the blue line) is positioned at approximately 900 on the y-axis in the Scan
window. For subsequent runs, the PMT gain will need to be adjusted only if the
y-axis value is out of the range of 800–1000 on the y-axis. Different PMT gains are
required for the three run modes (XL Scan, Full Scan, and BaseSprinter).
To check the PMT gain:
1.
While the Plate Check is running, view the four base lines (each line
represents one of the four filters) in the Scan window of the 373XL
collection program.
2.
Position the cursor so the tip of the arrow just touches a flat section of the
lowest scan line (usually the blue line), as in Figure 4-12.
Read y-axis here
Figure 4-12. Cursor positioned correctly to measure PMT gain
3.
Read the y-axis value displayed in the upper-left hand corner of the scan
window.
• If the lowest scan line is positioned between 800–1000 on the y-axis,
cancel the Plate Check, and then proceed to Inserting the Sharks-tooth
Comb on page 4-34.
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Applied Biosystems
• If the lowest scan line is outside the recommended range, adjust the
PMT gain.
To adjust the PMT gain:
1.
While the Plate Check is running, choose Manual Control from the
Window menu.
2.
Adjust the size and position of the Scan and Manual Control windows so
you can see both windows without any overlap.
3.
In the Manual Control window, choose PMT Gain from the Fxn Name
pop-up menu.
4.
Type a new value, then click Execute.
Note
If you enter a larger number than the one displayed, the lines in the
Scan window will shift up; a smaller number causes them to shift down.
5.
Check the y-axis value in the Scan window. (First click on the Scan window
to make it active.)
6.
Repeat steps 3–5 until the PMT gain is within the recommended range.
7.
Close the Manual Control window.
8.
Cancel the Plate Check in the Run window.
Note
After selecting a different run mode, you must change the PMT gain.
Inserting the Sharks-tooth Comb
Note
If you are using a square-tooth comb, skip this section and proceed to
Completing Instrument Setup on page 4-36.
To insert the sharks-tooth comb:
1.
Insert the sharks-tooth comb (see Figure 4-13).
a.
4-34
Center the sharks-tooth comb in the glass notch and carefully slide it
4 Using the Instrument in GeneScan Mode
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Applied Biosystems
between the glass plates. Make sure all of the points on the comb
enter the space between the plates at the same time, to avoid bending
or breaking the teeth. Carefully align the center registration line with
the registration mark on the plates.
b.
Slide the comb down until the tips of all the teeth just touch or slightly
depress the surface of the gel. Do not deform the wells. Make sure the
teeth just barely indent the well surface. All of the teeth must touch
the surface of the gel. If the surface of the gel is not completely flat in
the loading region, some of the teeth may have to be inserted below
the surface of the gel (up to 0.5 mm), so all of the teeth touch the gel
surface. If a tooth has penetrated the gel surface, do not attempt to
withdraw the comb, or sample will leak into adjacent wells.
Figure 4-13. Sharks-tooth comb just touching the gel
March 2001
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Applied Biosystems
Completing Instrument Setup
After completing Plate Check, do the following:
Classic
1.
If you are using a square-tooth comb, attach the D-clamps to the upper buffer
chamber, if they are not already attached.
a.
Remove the screws at either side of the upper buffer chamber.
b.
Place D-clamps so that the screw holes are aligned.
c.
Insert the screws and turn them a few times.
2.
Place the upper buffer chamber at the top of the gel plates by placing it on
the upper shelf (Figure 4-3 on page 4-12) and sliding the front brackets of
the chamber over the glass plates. The brackets should sit over the spacers
on the outside edges of the plate.
3.
For square-tooth comb runs, clamp the glass plates to the upper buffer
chamber by placing the D-clamp against the front of the glass plates and
inside the brackets of the buffer chamber.
The brace is not used for square-tooth comb runs.
4.
For sharks-tooth runs, put the alignment brace in place against the front of
the glass plates, inside the brackets of the buffer chamber, and resting on
top of the heat plate.
The alignment brace is made of highly polished acrylic so the numbers and
the comb are readily visible through the plastic. Do not mark the
alignment brace with felt-tipped pens, as the use of most organic solvents
to remove the ink will dull the surface of the brace and reduce its clarity.
Some ink may also fluoresce.
5.
Note
4-36
Tighten the screws on the buffer chamber equally on both sides to
maintain even pressure across the D-clamp or alignment brace. Turn the
screws until you see the gasket compress completely against the glass.
Finger-tighten only—do not overtighten. You may need to apply light
pressure on the top of the brace to prevent it from moving during
tightening.
The D-clamps are used in place of the alignment brace to prevent
deformation of the wells formed by the acrylamide square teeth.
4 Using the Instrument in GeneScan Mode
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Applied Biosystems
6.
Fill the upper buffer chamber with the 1X TBE buffer prepared earlier to
at least half-way up the notched part of the plate (~ 1.3 cm). Check for
leaks. Close the lid of the buffer chamber.
Note
If there is a leak, tighten the brackets slightly. If the leak continues,
siphon the upper buffer, then clean the gasket area on the chamber and
the area of the plate that contacts the gasket. Reassemble as before.
7.
Carefully fill the lower buffer chamber with 1X TBE buffer to about
two-thirds full.
Note
Leon
8.
Attach the top and bottom electrode leads located on the left side of the
electrophoresis chamber and close the door gently.
1.
If you are using a square-tooth comb, attach the D-clamps to the upper buffer
chamber, if they are not already attached.
2.
March 2001
Avoid splashing buffer against the back of the chamber since it will spill
over into the liner. Remove any bubbles that are under the bottom edge
of the gel plates using a clean needle and syringe. Bend the needle at
an angle to guide the bubbles out.
a.
Remove the screws at either side of the upper buffer chamber.
b.
Place D-clamps so that the screw holes are aligned.
c.
Insert the screws and turn them a few times.
For 24-cm runs only, slide the heat plate into place against the glass plates, as
follows (see Figure 4-4 on page 4-13):
a.
Rest the lower edge of the heat plate (the edge with the supports
protruding on both sides) on the upper inside ledge of the beam-stop
bar where it meets the glass plate. The supports rest in the notches.
b.
Stand the heat plate vertically against the glass plates.
c.
Secure the beam-stop swivel clamps.
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Applied Biosystems
3.
Place the upper buffer chamber at the top of the gel plates, on the
supports. Slide the clamps over the plates.
4.
For square-tooth comb runs, clamp the glass plates to the upper buffer
chamber by placing the D-clamps against the front of the glass plates and
inside the brackets of the buffer chamber.
The brace is not used for square-tooth comb runs.
5.
For sharks-tooth runs, put the alignment brace in place against the front of
the glass plates, inside the brackets of the buffer chamber, and resting on
top of the heat plate.
The alignment brace is made of highly polished acrylic so the numbers and
the comb are readily visible through the plastic. Do not mark the
alignment brace with felt-tipped pens, as the use of most organic solvents
to remove the ink will dull the surface of the brace and reduce its clarity.
Some ink may also fluoresce.
Note
6.
Note
7.
To use the D-clamps with 24-cm WTR, you must omit the heat plate.
Tighten the screws on both ends of the upper buffer chamber equally to
maintain even pressure across the alignment brace or D-clamps. Turn the
screws just until you see the gasket compress completely against the
glass—do not overtighten.
The D-clamps are used in place of the alignment brace to prevent
deformation of the wells formed by the acrylamide square teeth.
Fill the upper buffer chamber with the 1X TBE buffer prepared earlier to
at least 2/3 up the notched part of the plate (greater than 1.3 cm). Check
for leaks. Close the lid of the buffer chamber.
If there is a leak, slightly tighten the brackets. If the leak continues, siphon
the buffer out of the upper buffer chamber and clean the gasket area on
the chamber. Also, clean the area of the plate that contacts the gasket.
Reassemble as before.
8.
4-38
Carefully fill the lower buffer chamber with 1X TBE buffer to about
two-thirds full. Avoid splashing buffer against the back of the chamber
4 Using the Instrument in GeneScan Mode
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Applied Biosystems
because it will spill over into the liner. Bubbles should be removed if they
are under the bottom edge of the gel plates. This can be done with a clean
needle and syringe, with the needle bent at an angle to guide the bubbles
out.
Stretch
9.
Attach the top and bottom electrode leads located on the left side of the
electrophoresis chamber and gently close the door.
1.
If you are using a square-tooth comb, attach the D-clamps to the upper buffer
chamber, if they are not already attached.
1.
a.
Remove the screws at either side of the upper buffer chamber.
b.
Place D-clamps so that the screw holes are aligned.
c.
Insert the screws and turn them a few times.
Place the upper buffer chamber at the top of the gel plates, on the white
plastic shelf. Slide the clamps over the plates but do not tighten the clamps.
(To ensure a tight seal between the buffer chamber and the glass plates,
make sure the gasket on the upper buffer chamber is dry.)
IMPORTANT
2.
March 2001
If you have two different upper buffer chambers, use the larger of the
two for 34 cm runs. Use the small upper buffer chamber for all other
GeneScan runs.
Follow the instructions for the WTR and comb configuration you are
using:
•
For 24-cm and 34-cm runs with a sharkstooth comb: Slide the plain acrylic
alignment brace over the comb, between the upper buffer chamber clamps
and the glass plates. Finger-tighten the clamps; be careful not to over
tighten.
•
For 24-cm and 34--cm runs with a square-tooth comb: Tighten the screws on the
D-clamps; be careful not to over tighten.
•
For 6-cm and 12-cm runs with a square-tooth comb: Slide the acrylic alignment
brace with the temperature sensor over the comb, between the upper
buffer chamber clamps and the glass plates. Finger-tighten the clamps; be
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Applied Biosystems
careful not to over tighten. Plug the temperature sensor into the socket at
the top left of the chamber.
Note
You must have the temperature sensor from either a heat plate or the
acrylic brace connected for the instrument to run.
The alignment brace is made of highly polished plastic so the numbers and
the comb are readily visible through the clear plastic. Do not mark the
alignment brace or glass plates with felt-tipped pens, as the use of most
organic solvents to remove the ink will dull the surface reduce its clarity.
Some ink may also fluoresce.
3.
Plug the electrode lead on the upper buffer chamber into one of the two
jacks. For 34 cm gels, use the top (rear) socket, and for all other gels, use
the lower (front) socket (see Figure 4-5 on page 4-15).
4.
Fill the upper buffer chamber to at least two-thirds up the notched part of
the plate (greater than 1.3 cm). Check for leaks. Close the lid of the buffer
chamber.
If there is a leak, slightly tighten the brackets. If the leak continues, siphon
the buffer out of the upper buffer chamber and clean the gasket area on
the chamber. Also, clean the area of the plate that contacts the gasket.
Reassemble as before.
5.
Carefully fill the lower buffer chamber with 1X TBE buffer to about
two-thirds full. For 34 cm runs, fill the lower buffer chamber until the
bottom of the glass plates is covered. Avoid splashing buffer against the
back of the chamber because it will spill over into the liner. Bubbles should
be removed if they are under the bottom edge of the gel plates. This can
be done with a clean needle and syringe, with the needle bent at an angle
to guide the bubbles out.
IMPORTANT
6.
4-40
The buffer must completely cover the bottom of the gel plates. You will
need to prepare 2 L of buffer for 34 cm gels. For all other lengths, the
standard volume of 1.5 L is sufficient.
Attach bottom electrode lead located on the left side of the electrophoresis
chamber.
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Applied Biosystems
Preparing and Loading Samples
Pre-running the Gel
Pre-run the gel for 5 minutes to make sure all connections and components of the
electrophoresis system are working properly.
To pre-run the gel:
1.
Flush all of the wells with running buffer. Direct the needle of a syringe
filled with running buffer at the space between adjacent comb teeth, and
gently flush the well.
Remove any bubbles under the bottom edge of the gel plates in the lower
buffer chamber. Use a clean needle and syringe, with the needle bent at an
angle to guide the bubbles out.
2.
If you have not done so, choose the PreRun module from the PreRun
pop-up menu.
3.
Click the PreRun button.
The instrument performs electrophoresis without collecting data during a
pre-run.
March 2001
4.
Pre-run the gel for 5 minutes.
5.
Click Cancel in the Run window.
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Applied Biosystems
Preparing Samples for Electrophoresis
Add your samples to loading cocktail, as described here. After the pre-run
(~5 minutes), load the samples and start the run.
WARNING
CHEMICAL HAZARD. Formamide is a known teratogen and can
cause birth defects. Before using formamide, read the MSDS
found in the Safety Summary. Wear protective eyewear, gloves,
and safety clothing when working with formamide. Wash
thoroughly after handling formamide.
To prepare samples for electrophoresis:
1.
Prepare loading cocktail by mixing (5:1:1)
• 2.5 µL deionized formamide
• 0.5 µL Blue Dextran EDTA [1 mg Blue Dextran per 1 mL EDTA
(25–50 mM, pH 8.0)]
• 0.5 µL appropriate GeneScan Size Standard
Store at 4° C for up to 1 week.
2.
Determine the appropriate dilution of your fluorescently-labeled PCR
products.
The dilution used is dependent on the volume of your PCR reaction
labeling strategy, and efficiency of label incorporation.
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Applied Biosystems
3.
Prepare sample for loading.
Use the following table to determine the appropriate sample, cocktail, and
loading volumes for the comb you are using.
Table 4-2. Sample, Cocktail, and Loading Volumes
Comb Configuration (number of wells)
24
34/36
50
66
Sample volume* (µL)
1.5
1.5
1.5
1.5
Cocktail volume (µL)
3.5
3.5
2.5
2.5
Loading volume (µL)
5
4
1.5–2.5
0.5–1.5
*User must determine the correct concentration, such that the signal is not >2000 fluorescent units.
Note
In the 66-well configuration, the best resolution is achieved with 0.5 µL
load volumes. It might be necessary to concentrate your PCR product
by vacuum drying or evaporation. Perform concentration before adding
the loading cocktail.
4.
Vortex and centrifuge briefly.
5.
Heat samples at 95°C for 5 minutes.
6.
Immediately place samples on ice until ready to load.
Loading the Samples and Starting the Run
IMPORTANT
Be sure you load samples in the lanes specified in the Run window. The
sample information in the Run window when you start the run is the
information stored in the Gel file. Changes you make in the Run window
after you start the run are not implemented.
For GeneScan applications, we recommend you use a square-tooth comb.
Sharks-tooth combs may be used only if there is a vacant lane between samples.
Loading every other lane for a sharks-tooth prevents even the smallest amount of
sample bleed-through from samples in adjacent lanes and is imperative for
GeneScan analysis accuracy.
March 2001
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Applied Biosystems
Note
If you are doing GeneScan analysis and have formed the wells using
the square tooth comb, you can load the samples in consecutive lanes.
Whenever possible, load a sample in lane one to help orient the tracker during
analysis. For best results, follow the procedure described here.
Note
In the following procedures, each button in the Run window has a
corresponding menu choice in the Instrument menu. You can either
click the button in the Run window or choose the command from the
Instrument menu.
Make sure you deliver the samples to the bottom of the wells. Use either a singleor a multi-channel pipetter/syringe equipped with a flat tip less than 0.3 mm in
diameter (we recommend one with a diameter of 0.17 to 0.20 mm). Do not
overload the lanes. Follow the recommendations in Table 3-3 on page 3-40 for the
allowable loading volumes. If you use too much sample volume or quantity, you
will experience poor resolution.
To load samples from a square-tooth comb:
1.
If you are pre-running the gel, click Cancel in the Run window.
2.
Carefully flush all of the wells with running buffer.
Tip
3.
Place formamide or loading buffer in the well to the left of your first
sample lane, and in the well to the right of your last sample lane (the
comb creates wells to either side of the numbered wells). The
formamide in the buffer helps focus the bands in the first and last lanes.
If you are loading a square tooth comb for GeneScan analysis, you can load
the wells consecutively.
IMPORTANT
4-44
Whenever possible, load a sample in lane one. Avoid skipping more
than one lane between samples. It is better to leave extra empty lanes
at the right end of the gel. The lane tracker may not work properly if
there are many empty lanes between samples.
4 Using the Instrument in GeneScan Mode
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Applied Biosystems
4.
Click Run to start the run.
A dialog box appears, allowing you to change the name and location of the
gel file created by the run.
5.
Click OK when you are satisfied with the name and location for the gel file.
Note
If a dialog box appears indicating you do not have enough room on your
hard disk to save the files created by a run, delete files (particularly Gel
files) to create enough space, making sure to back them up on a floppy
disk or other storage device first, then click Run again.
To load samples from a sharks-tooth comb:
1.
If you are pre-running the gel, click Cancel in the Run window.
2.
Carefully flush all of the wells with running buffer.
Tip
3.
Place formamide or loading buffer in the well to the left of your first
sample lane, and in the well to the right of your last sample lane (the
comb creates wells to either side of the numbered wells). The
formamide in the buffer helps focus the bands in the first and last lanes.
Immediately load the sample into each of the odd-numbered wells.
If you are loading fewer than half the samples allowed by the comb, skip
steps 6–10 and perform step 11. If you are loading a square tooth comb,
you can load the wells consecutively and skip steps 4–9.
IMPORTANT
4.
Whenever possible, load a sample in lane one. Avoid skipping more
than one lane between samples. It is better to leave extra empty lanes
at the right end of the gel. The lane tracker may not work properly if
there are many empty lanes between samples.
Click Run in the Run window to start the run.
A dialog box appears, allowing you to change the name and location of the
gel file created by the run.
March 2001
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Applied Biosystems
5.
Click OK when you are satisfied with the name and location for the gel file.
Note
If a dialog box appears indicating you do not have enough room on your
hard disk to save the files created by a run, delete files (particularly Gel
files) to create enough space, making sure to back them up on a floppy
disk or other storage device first, then click Run again.
6.
Choose Status from the Window menu to verify that electrophoresis and
scanning resume (refer to page 5-50). If either electrophoresis or scanning
does not resume, cancel the run and restart it.
Figure 4-14 summarizes the buttons you click in the Run window from the time
you start the plate check for a typical run. They are listed from top to bottom in
the order you use them.
Note
4-46
The tasks described in Figure 4-14 are detailed in the preceding pages.
4 Using the Instrument in GeneScan Mode
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Applied Biosystems
Button to click
Module in effect
Task to perform
Plate Check module
Check plates and gel for
fluorescence
n/a
Pre-run module
(Stops plate check scan)
Add buffer and check for leaks
Mount front heat-transfer plate
(Electrophoreses gel without
data collection to equilibrate
temperature)
Prepare samples while
pre-running
Pre-run module
Load first set of samples during
pause
Pre-run module
(Electrophoreses samples into
gel without data collection)
Pre-run module
Load second set of samples
during pause
n/a
(Stops pre-run)
Run module
(Starts electrophoresis and
data collection)
Figure 4-14. Run window buttons used during a typical run
March 2001
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Observing Instrument Status and Data
After you begin electrophoresis and data collection, data appears on the
computer screen as it is collected. You can open up to four windows to view the
data and the instrument status. Only one window can be active at any given time,
but status windows still update when they are not active.
Note
If they are displayed, status windows continually update during a run.
They do so even when another application is running.
Open the data collection and status windows by choosing the name of the window
of interest from the Window menu.
Data Collection Status Windows
To observe instrument status, choose one of the following commands from the
Window menu. The page reference next to a window name indicates where you
can find detailed information about that window.
•
Status (refer to page 5-50)
•
Electrophoresis History (refer to page 5-57)
•
Log (refer to page 5-57)
Note
4-48
After the run is completed, you can choose Log from the Window menu
to display a comprehensive record of all error and status messages
generated during the run.
4 Using the Instrument in GeneScan Mode
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Applied Biosystems
Real-time Data Windows
The data displayed in the real-time data windows (the Scan window and the Gel
window) represents light intensities collected by the data collection software from
the filter wheel and PMT in the instrument. The colors in the raw data windows
represent the wavelengths of the dyes being detected, rather than the bases being
detected. This is explained in The Electrophoresis History Window on page 5-57.
To observe the data as it is being generated, choose one of the following from the
Window menu:
March 2001
•
Scan (refer to page 5-55)
•
Gel (refer to page 5-56)
4 Using the Instrument in GeneScan Mode
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Applied Biosystems
Cleaning Up After the Run
After your run is complete, perform the following general cleanup steps:
•
Remove the gel from the instrument.
•
Clean the plates, comb, and spacers.
Removing the Gel from the Instrument
Classic
and
Leon
1.
Disconnect the electrode leads from both buffer chambers.
2.
Unlatch the beam-stop bar and pull it forward.
3.
Carefully remove the upper buffer chamber, glass plates, and heat plate
(Leon only) as one unit. Discard the buffer.
You can also set up a siphon system to drain the upper and lower buffer
chambers before removing them.
Stretch
4.
Remove the lower buffer chamber and discard the buffer.
5.
Clean up any liquid left in the electrophoresis chamber.
6.
Unclamp the plates from the upper buffer chamber.
7.
Rinse the buffer chambers with dH2O and allow them to air dry.
1.
Disconnect the electrode leads from both buffer chambers.
2.
Unlatch the beam-stop bar and pull it forward.
3.
Carefully remove the upper buffer chamber and glass plates as one unit.
Discard the buffer.
4.
Remove some of the buffer from the lower buffer chamber before
removing it.
This is especially important for 34-cm runs, as the buffer level is very high.
You can also set up a siphon system to drain the upper and lower buffer
chambers before removing them.
5.
4-50
Remove the lower buffer chamber and discard any remaining buffer.
4 Using the Instrument in GeneScan Mode
March 2001
Applied Biosystems
6.
Clean up any liquid left in the electrophoresis chamber.
7.
If you plan to change plate length next time, unplug the temperature
sensor, and then remove the heat plate, otherwise leave both in place.
8.
Unclamp the plates from the upper buffer chamber.
Note
9.
If the unnotched plate has been silated with a bind-silane compound,
use 0.1 N HCl or NaOH to remove the gel from the silated area, or
scrape it off gently.
Rinse the buffer chambers with dH2O and allow them to air dry.
Cleaning the Plates, Comb, and Spacers
March 2001
1.
Gently push a plastic wedge almost all the way in at the bottom of the plates
(avoid the top notches) and pry the plates apart. Do not use too much
force or the plates might chip. Remove the sharks-tooth comb and gel
spacers.
2.
Lay a large Kimwipe on the gel and roll it up, lifting the gel off the plate,
and discard appropriately.
3.
Rinse the plates with water. Rub with a gloved hand to dislodge remaining
gel bits, then rinse again with water.
4.
Wash with a detergent such as Alconox that will not leave a residue, rinse
with dH2O, and allow to dry.
5.
Clean the comb and spacers and store them.
4 Using the Instrument in GeneScan Mode
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Applied Biosystems
WARNING
4-52
CHEMICAL HAZARD. The acrylamide and bis-acrylamide used to
prepare sequencing gels for the ABI PRISM 373 DNA Sequencer
with XL Upgrade are toxic chemicals whose use requires utmost
attention to safety. Be sure to read the Material Safety Data Sheets
(MSDSs)for emergency and first-aid procedures. MSDSs are in the
ABI PRISM 373 DNA Sequencer with XL Upgrade Pre-Installation
Manual, P/N 901173. Acrylamide is a neurotoxin that can be
absorbed through the skin. Wear a laboratory coat, eye protection,
and gloves. The handling and disposal of chemical waste is the
responsibility of the user, and not the Applied Biosystems service
representative.
4 Using the Instrument in GeneScan Mode
March 2001
Applied Biosystems
Analyzing the Data
If you choose Auto Analyze for your samples in the GeneScan Run window, the
data collected on the ABI PRISM 373XL is automatically analyzed by the GeneScan
Analysis program. For complete instructions on using the analysis program, refer
to the ABI PRISM GeneScan Analysis Software User’s Manual.
Automatic data analysis occurs approximately 1 to 10 hours after you start data
collection (the amount of time depends on the run conditions and WTR you
chose). Three channels of data are analyzed for each lane containing a sample on
the gel. The analysis software prints electropherograms (if you selected AutoPrint
in the Sample Sheet) and displays the gel window on the computer screen.
March 2001
4 Using the Instrument in GeneScan Mode
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Applied Biosystems
Evaluation Checklist
Before evaluating the quality of the data in your Samples files, check the following:
4-54
•
After analysis, always view the Gel file on the computer screen. Check
tracking and look for any atypical characteristics of the gel that may
indicate a problem. For more information on fine tuning the gel file, refer
to the ABI PRISM GeneScan Analysis Software User’s Manual.
•
Examine data obtained with control reactions. If you obtain good data with
control reactions, you can eliminate the possibility of problems with
reagents, equipment, or general technique.
•
Study the analyzed data. Take note of the peak shape, the amount of noise,
any obscured, crowded peaks, or other peaks that appear under peaks.
•
Review Appendix F for strategies you can employ to optimize PCR
performance.
•
For forensics applications, run a positive control of the analyst’s DNA to
check for possible contamination of unknown samples and a negative
control.
•
Examine the standard curve for the sizing standard in each lane. Ensure
the curve is linear and the proper peaks have been identified. If not,
reanalyze the lane defining a new standards file for that lane. Refer to the
ABI PRISM GeneScan Analysis Software User’s Manual.
4 Using the Instrument in GeneScan Mode
March 2001
Applied Biosystems
Characteristics of Good Data
An electropherogram from a Sample file that contains good data has sharp,
well-defined peaks.
Figure 4-15. Example of good data with sharp, well-defined peaks
March 2001
4 Using the Instrument in GeneScan Mode
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Applied Biosystems
Characteristics of Poor Data
No Peaks
The absence of peaks in your results file could be due to no PCR amplification.
To determine if this is the case, run another gel and include a lane of Taq
Standard PCR Control or another reaction you know that has run successfully
before. If you have no bands in your sample, but the size standard appears to be
good, there was no amplification. For tips on optimizing PCR, see Appendix E.
Too Many Peaks— Noisy Data
If artifact bands, extra peaks, or peaks under peaks appear in your Sample files,
try one or several of the following:
4-56
•
Check for possible PCR carryover.
•
Check PCR technique for practices that may have led to sample
contamination.
•
Check primer sequences for 3' complementarity.
•
Design longer primers.
•
Increase the amount of target DNA.
•
Reduce the primer concentration.
•
Reduce the number of cycles.
•
Raise the annealing temperature.
•
Decrease MgCl2 concentration in your reaction.
4 Using the Instrument in GeneScan Mode
March 2001
Applied Biosystems
Smeared or Fuzzy Bands
If you see smeared or fuzzy bands, try one or several of the following:
March 2001
•
Reduce the concentration of AmpliTaq DNA Polymerase.
•
Create a Master Mix of PCR reagents to ensure each reaction is getting the
correct amount of all reagents.
•
Increase the annealing temperature and consider a two-temperature PCR
protocol.
•
Reduce the magnesium concentration.
•
Minimize the incubation times of the annealing and extension steps.
•
Decrease the number of cycles.
•
Perform electrophoresis at lower voltage.
4 Using the Instrument in GeneScan Mode
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Applied Biosystems
Troubleshooting Table
Table 4-3. Troubleshooting for PCR and Electrophoresis Conditions
Observation
Possible Causes
Potential Solution
PCR Conditions
Unsuccessful
amplification
(faint or no
peaks).
4-58
Impure DNA template. Try different extraction procedures combined
with filtration of DNA through a spin column
before amplification.
PCR inhibitors may
exist in the DNA
sample.
Insufficient template
DNA for human
identification
experiments.
Use recommended amount of template DNA.
Use a procedure such as QuantiBlot™
Human DNA Quantitation Kit to accurately
quantitate human DNA samples.
If DNA is stored in water, check water purity.
Insufficient enzyme
activity.
Use the recommended amount of AmpliTaq
DNA Polymerase.
MgCl2 solution not
added to PCR control
DNA reaction.
Add appropriate amount of
MgCl2 solution to amplification reaction.
Incorrect thermal
cycling parameters.
Check protocol for correct
thermal cycling parameters.
GeneAmp PCR
instrument system
failure.
Choose correct amplification program for
each primer pair.
4 Using the Instrument in GeneScan Mode
March 2001
Applied Biosystems
Observation
Possible Causes
Potential Solution
Unsuccessful
amplification
(faint or no
peaks).
High salt
concentrations of K+,
Na+, or Mg2+.
Incorrect volume of DNA can alter pH: use
correct amount of DNA, and buffer.
High concentration of
EDTA.
Incorrect pH.
Unsuccessful
amplification
(faint or no
peaks).
Weak signal.
March 2001
Tubes not seated
tightly in the
thermal cycler during
amplification.
Push reaction tubes firmly into contact with
block before first cycle.
Wrong style tube.
Use Applied Biosystems GeneAmp
Thin-Walled Reaction Tubes for the DNA
Thermal Cycler 480, or MicroAmp Reaction
Tubes with Cap for the GeneAmp PCR System 9600.
Primer concentration
too low.
Use recommended primer concentration.
For multiplex PCR,
lack of 2 minute down
ramp time between
denaturation and
annealing.
Between denaturation and annealing, add 2
minute down ramp time to thermal cycling
program. For multiplex reactions, the fastest
ramp time is not necessarily the best.
4 Using the Instrument in GeneScan Mode
4-59
Applied Biosystems
Observation
Possible Causes
Potential Solution
Weak signal.
Incorrect Mg2+
concentration.
Store DNA in H2O. The DNA volume should
not be more than one fifth of the total reaction
volume. EDTA in TE storage buffer affects
the MgCl2 concentration.
Add correct amount of Mg2+ to reaction mix.
Use correct dilution (1X) of 10X PCR buffer.
Check dilution of dNTP stocks concentrations
higher than recommended may lower the free
Mg2+.
Perform additional organic extractions with
EtOH precipitations and /or filter DNA
through a spin column before amplification to
remove any potential PCR inhibitors.
Inconsistent
results with
control DNA.
4-60
Incorrect PCR thermal
profile program.
Choose correct temperature control parameters for PCR thermal profile (See GeneAmp
PCR System 9600 User’s Manual).
System 9600
misaligned lid.
Align 9600 lid properly so that white stripes
align after twisting the top portion clockwise.
For DNA Thermal
Cycler 480, Improper
tube placement in
block.
Refer to DNA Thermal Cycler 480 User’s
Manual.
Pipetting errors.
Calibrate pipettes, attach tips firmly, check
technique. Make a master mix of reagents.
Combined reagents
not spun to bottom of
tube.
Place all reagents in apex of tube, spin briefly
after combining.
Combined reagents
left at room
temperature or on ice
for extended
periods of time.
Put tubes in block immediately after reagents
are combined.
4 Using the Instrument in GeneScan Mode
March 2001
Applied Biosystems
Observation
Possible Causes
Potential Solution
Unbalanced
signals with
control DNA.
Pipetting errors.
Calibrate pipets, attach tips firmly, check
technique.
Unbalanced
signals for
samples.
PCR inhibitors may be
present in DNA
sample.
Try different extraction
procedures. Filter DNA through a spin
column before amplification.
Extra peaks
visible when
sample(s) is
known to
contain DNA
from a single
source.
Contamination with
exogenous DNA.
Use appropriate techniques to avoid introducing foreign DNA during laboratory
handling.
Renaturation of
denatured samples.
Load sample immediately
following denaturation, or store on ice until
you are ready to load.
Too much DNA in
reaction, promoting
renaturation.
Use recommended amount
of template DNA. Use a procedure such as
QuantiBlot Human DNA
Quantitation Kit to accurately quantitate DNA
sample.
Too much DNA
amplified and/or
loaded resulting in
crossover between
color channels.
Check if any peak, particularly a homozygote,
is over 6000 peak height. Re-run PCR using
less DNA, or load less sample during
electrophoresis.
During thermal
cycling, temperature
ramps too quickly.
For GeneAmp instrument
system, program one minute ramp rates.
For StockMarks For Cattle protocol,
weak signal for
TGLA53.
March 2001
4 Using the Instrument in GeneScan Mode
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Applied Biosystems
Observation
Possible Causes
Potential Solution
Peaks are split
into two peaks
that are 1 bp
different in size.
Partial non-template
addition of an extra
nucleotide to the blunt
end of the PCR
product. High Mg2+
concentrations can
lower the degree of
nucleotide addition.
Add correct amount of Mg2+ to reaction mix.
Signal
continually gets
weaker.
Outdated or
mishandled reagents.
Check expiration dates on
reagents and store and use
according to manufacturers
instructions.
Compare with fresh reagents.
Electrophoresis Conditions
Inconsistent
sizing of known
DNA sample.
Inadvertent change in
analysis parameters.
Check settings for GeneScan analysis
parameters.
Incorrect internal
standard.
Check internal size standard peaks against
definition used.
Incorrect gel
composition.
Check if gel composition matches protocol
requirements.
Check urea concentration.
Data was not
automatically
analyzed.
4-62
Incorrect sample
sheet.
Check Sample Sheet selection in data
collection program, or check sample sheet
embedded in Gel file.
Sample sheet not
completed.
Correctly complete sample sheet.
4 Using the Instrument in GeneScan Mode
March 2001
Applied Biosystems
Observation
Possible Causes
Potential Solution
Crossover of
one or more
colors different
than the
primary color.
Incorrect matrix
chosen.
Check matrix selection in Run window of data
collection program or Gel file.
Sample overloaded.
Load sample volume recommended in
application protocol.
Samples run
faster than
usual with
decreased
resolution.
Incorrect gel
composition.
Check whether gel composition matches
protocol requirements.
Continued
weakening of
signal over
time.
Degraded primers.
Store primers at -20˚C when not in use. Keep
on ice during set-up.
Off-scale data not
Additional
colors displayed matrixed correctly.
underneath the
position of one
strong peak.
March 2001
Repeat electrophoresis; load less sample.
Peaks
appearing in a
dye color that
should not be
present.
Bleed through from
other colors due to
off-scale data.
Repeat electrophoresis; load less sample.
Allele peaks
seen in
correct
molecular
weight range,
with additional
peaks seen
outside this
range.
Bleed through from
other colors due to
off-scale data.
Primers not
optimized.
Repeat electrophoresis; load less sample.
Refer to Appendix E.
4 Using the Instrument in GeneScan Mode
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Applied Biosystems
4-64
Observation
Possible Causes
Potential Solution
With allele
peaks of high
intensity,
GeneScan
sites many
small peaks.
Adjust minimum peak height; reanalyze
Background above
minimum peak height. (See the GeneScan Analysis Software User’s
Manual).
No current.
Incorrect TBE buffer
concentrations.
Check concentrations of TBE buffer:
10X in gel
1X in running buffer.
Instrument not
assembled correctly.
Check if leads are secure, and make sure
that the bottom and top of gel immersed in
running buffer.
Instrument parts
broken.
Check for breaks in leads,
buffer chambers, or platinum wire.
Repeat electrophoresis; load less sample.
Too much PCR
product loaded on gel.
4 Using the Instrument in GeneScan Mode
March 2001
Applied Biosystems
Archiving the Data
For best performance, keep as few files as possible on your hard disk. Do not
archive gel files and run folders on your hard disk. It is important that you copy
all files you want to save onto a backup medium before beginning another
sequencing run, both to preserve the files and to free disk space.
Files Created During a Run
When you set up a run, the data collection software creates a Run folder and a Run
file. During the run, the program automatically creates and saves two files: the Gel
file and the Run Log file. After the run, the analysis program creates Sample files.
Run Files
Each Run file stores information associating specific samples with specific lane
positions. It also stores information about the Sample Sheet and module
associated with the run. Run files are small enough to store on floppy disk.
Gel Files
Gel files contain the raw data acquired by the data collection program and
typically take up 30–50MB of disk space. A separate Gel file is created in the Run
folder each time you perform a run. Because Gel files take a substantial amount
of space, you should delete them from your hard disk after you have obtained
satisfactory Sample files. You do not usually need to keep gel files once the lane
tracking is verified and sample files are created.
Use magnetic tapes, removable cartridge drives, or optical drives to archive gel
files. Gel files are too large to fit on floppy disks.
IMPORTANT
Do not discard any gel file until you have verified the tracking and taken
corrective action if necessary.
Run Log Files
Run logs provide a good record of your runs. Individually, Run Log files are small
enough to archive on floppy disks.
March 2001
4 Using the Instrument in GeneScan Mode
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Applied Biosystems
Sample Files
Use floppy disks, a magnetic tape drive, a removable cartridge drive, or an optical
drive to archive sample files. A 1.4 MB, high-density disk holds about six Sample
or Sequence files. A Sample file is 150–200KB in size, depending on the length of
the sequencing run. Save a Sample file when you feel confident that the channel
selections (tracking) are correct for the sample.
Other Files
Other files provide information for a run that you might have customized and
should therefore save. These include the Sample Sheet, analysis settings, or matrix
files you create.
Sample Sheet Files
You can reuse the Sample Sheet, and it is a good reference after data collection.
Individually, Sample Sheet files are small enough to archive on floppy disks.
Custom Analysis Settings and Matrix Files
If you have created customized files, you should keep backup copies of the files on
a separate medium. Each of these types of files alone is small enough to store on
a floppy disk.
4-66
4 Using the Instrument in GeneScan Mode
March 2001
400 I'M INVISIBLE
Applied Biosystems
5 Using the Data Collection Software
Contents
Overview of Data Collection Menu Commands
Apple Menu
File Menu
Edit Menu
Instrument Menu
Window Menu
Using the Data Collection Program
Starting the Data Collection Program
Setting Preferences
Setting Folder and File Preferences
Setting Folder Location Preferences
Setting File Name Preferences
Setting Defaults for Sequencing Analysis Applications
Setting Sequence Sample Sheet Defaults
Setting Sequence Run Defaults
Setting Defaults for GeneScan Analysis Applications
Setting GeneScan Sample Sheet Defaults
Setting GeneScan Run Defaults
General Settings
Setting Dye Indicators Defaults
Creating and Editing a Sample Sheet
Importing and Exporting Sample Sheet Information
Creating a Sequence Sample Sheet
Creating a GeneScan Analysis Sample Sheet
Editing a Sequence or GeneScan Analysis Sample Sheet
Using the Run Window
Opening the Run Window
Completing a Run Window
Starting a Pre-run or a Run
Starting from the Run Window
Starting from the Instrument Menu
March 2001
5 Using the Data Collection Software
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5-1
Applied Biosystems
Viewing the Data
Using the Data Collection Program Windows
Observing Instrument Status During and After a Run
The Status Window
The Log File
Viewing Data
Filters and Color of Data Display
The Scan Window
The Gel Window
The Electrophoresis History Window
Viewing Other Run-Related Information
The Sample Sheet
The Run File
Controlling the ABI PRISM 373 DNA Sequencer with XL Upgrade Manually
PMT Gain and Calibration File
Checking and Adjusting the PMT Gain
Recommended PMT Gain
Checking the PMT
Adjusting the PMT Gain
Making and Sending a Calibration File
Calibration File
Making a Calibration File
Sending a Calibration File
Saving and Printing Files
Saving Files
Archiving Files
Gel Files
Sample files
Printing Files
Setting Up the Page for Printing
Printing
Importing and Exporting Files
Exporting
Importing
Quitting the Data Collection Program
About Data Collection Program Files
5-2
5 Using the Data Collection Software
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March 2001
Applied Biosystems
ABI PRISM 373 DNA Sequencer with XL Upgrade Data Flow
Input and Output Files
Input Files
Output Files
Files Located in the System Folder
Temporary Files Created in the System Folder
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5 Using the Data Collection Software
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Applied Biosystems
5-4
5 Using the Data Collection Software
March 2001
Applied Biosystems
Overview of Data Collection Menu Commands
ABI PRISM 373 DNA Sequencer with XL Upgrade software programs are designed
with the easy-to-use pull-down menus and the point-and-click technique
commonly used in Macintosh-compatible programs. The menu bar at the top of
the screen gives access to all functions. You can also use keyboard commands in
many cases. If they are available, keyboard commands are listed opposite their
corresponding commands on the pull down menus.
This section briefly describes each of the four menus on the data collection
program main menu bar.
Apple Menu
At the left end of the menu bar is the Apple menu, a standard feature of
Macintosh applications. It displays desk accessories installed on your system.
The top command, About ABI PRISM 373 DNA Sequencer with XL Upgrade
Collection, opens the splash screen, which shows general information about the
data collection program. Click on it to close it.
Figure 5-1. Splash screen
March 2001
5 Using the Data Collection Software
5-5
Applied Biosystems
File Menu
The File menu contains commands for creating, opening, closing, and saving
windows, importing and exporting data to other file types, printing, and quitting
from the application.
Table 5-1. File Menu Commands
5-6
Command
Description
See for
Detail
Close, Save, Save
As..., Revert, Page
Setup..., Print..., Quit
Standard Macintosh system commands. See
the Macintosh System 7 Reference manual
Page 5-65
New... and Open...
Open Run files or Sample Sheets for
Sequencing or GeneScan applications. Use
New to create a new file, or use Open to open
an existing file.
Pages 5-34
to 5-40
Save a Copy In...
Saves a backup copy in a file other than the
original. The name of the file with which you
are working does not change.
Page 5-65
5 Using the Data Collection Software
March 2001
Applied Biosystems
Table 5-1. File Menu Commands (continued)
March 2001
Command
Description
See for
Detail
Import...
Imports text from files such as those generated
by a database into the grid of the Sample
Sheet or Run window.
Page 5-33
Export...
Exports data from the grid of the Sample Sheet
or Run window in tab-delimited text format for
database applications.
Page 5-33
Print One
Prints one copy of the active window,
bypassing the standard print dialog box.
Page 5-66
5 Using the Data Collection Software
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Applied Biosystems
Edit Menu
The Edit menu contains standard clipboard commands, commands for selecting
and searching, spreadsheet editing commands (Insert and Fill Down), and a
command for changing the time scale in the Electrophoresis History window.
Table 5-2. Edit Menu Commands
5-8
Command
Description
See for
Detail
Undo (Redo), Cut,
Copy, Paste, Clear,
and Show Clipboard
Standard Macintosh system commands. See
the Macintosh System 7 Reference manual
pages 5-32,
5-38
Select All
Selects all entries in the open window.
Fill Down
Applies a parameter change to all cells in a
column of a Sample Sheet or Run window.
Page 5-38
Set Scale...
Sets the scale of the panels in the
Electrophoresis History window.
Page 5-58
5 Using the Data Collection Software
March 2001
Applied Biosystems
Instrument Menu
The Instrument menu contains commands for starting, pausing and stopping the
ABI PRISM 373XL.
Table 5-3. Instrument Menu Commands
March 2001
Command
Description
Start Plate Check
Allows you to scan the gel and plates before loading Page 5-48
samples to ensure that no peaks are produced by
fluorescent particles on the glass plates or in the gel.
Start PreRun
Starts a pre-run of the gel, using parameters in the
pre-run module specified in the Run window.
Page 5-48
Start Run
Begins automatic execution of a run using
parameters in the run module specified in the Run
window.
Page 5-48
Pause
Temporarily interrupts a run. During a pause, no
additional functions are performed. The laser stops
scanning, but any data remaining in instrument
memory continues to be collected.
Page 5-48
Resume
Resumes a run that has been paused.
Page 5-48
Cancel Run
Only available if a module is currently running.
Allows you to immediately stop the current module
on the instrument. The run is ended and the Run file
is closed. You cannot resume the run.
Page 5-48
5 Using the Data Collection Software
See for
Detail
5-9
Applied Biosystems
Window Menu
The Window menu contains a list of the default windows: instrument Status, Log
file, Sample Sheet, Run file, Scan, Gel Image, and Electrophoresis History. At the
bottom it also contains a list of any other open windows. Choosing any window
from this menu brings that window to the front and makes it the active window.
• To move a window around on the screen, click the top border, hold the
mouse button down, and drag the window to another location on the
screen.
• To change the size of a window, click the size box in the lower right corner
of the window, hold the mouse button down, and drag the mouse to stretch
or shrink the window.
• To close a window, click the close box at the upper left corner of that
window or click in the window to make it active and choose Close (c-W)
from the File menu.
• To expand a window so that it displays the entire contents within the
window, click the zoom box in the upper right corner.
5-10
5 Using the Data Collection Software
March 2001
Applied Biosystems
Table 5-4. Window Menu
March 2001
Command
Description
See for
Detail
Status
Displays the status of hardware and status
of the run. During a run, it displays current
instrument conditions.
Page 5-50
Log
Text file that contains date- and
time-stamped status and error messages in
chronological order.
Page 5-51
Sample Sheet
Allows you to specify sample information for
each lane position in the gel. The
information you enter depends on the
analysis program you are using.
Page 5-58
Run
Allows you to specify the Sample Sheet and
module information for an automatic run.
Page 5-58
Scan
Displays electropherogram of raw data as it
is being collected during the run.
Page 5-55
Gel Image
Displays a reconstruction of actual data as it
would appear on an autoradiogram. It is
updated as data is collected from the
instrument.
Page 5-56
Electrophoresis History
Displays target and actual values for the
electrophoresis power supply and gel
temperature throughout the course of an
electrophoresis run.
Page 5-57
Manual Control
Displays a window for manual control of
certain functions during a run.
Page 5-59
Preferences...
Allows you to specify folder locations, file
name conventions, and default settings.
Page 5-16
5 Using the Data Collection Software
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Applied Biosystems
Using the Data Collection Program
Before you start an ABI PRISM 373XL run, the following conditions should apply:
• You have set or verified Preferences
• You have created the Sample Sheet and the Run file
• The instrument is ready and samples are loaded
• The Macintosh hard disk has adequate free space to store the collected
data (It needs a minimum of 40 to 60MB free disk space for an average
run.)
To learn about preparing gels and setting up the instrument, refer to Sections 2
and 3. For information about choosing a proper sequencing chemistry, refer to
the ABI PRISM DNA Sequencing Analysis Chemistry and Safety Guide. For information
about GeneScan experimental design, refer to the GeneScan 672 User’s Manual.
This section describes how to use the data collection software to create and edit
Sample Sheets and Run files, to perform a run, and to view the resulting data.
To prepare the software for a run, perform the following general steps:
• Restart the computer
• Start the data collection program
• Set or verify preferences
• Create a Sample Sheet*
• Create a Run file
When you start the data collection program and begin a run, the software displays
data as it is received from the ABI PRISM 373XL, and you can view the data in
different views, described on page 5-49. The data is stored on the computer.
The data collection program typically runs for approximately 3.5–16 hours
(1–6 hours for GeneScan) from the time you start the run to the time data analysis
begins.
* If
a Sample Sheet already contains information that applies to your samples, you do not need
to create a new one. Specify lane position for each sample when you create the Run file.
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Starting the Data Collection Program
• Choose Restart from the Special menu in the Finder to restart your
computer.
The data collection software should automatically open when you restart
the computer. If it does not, double-click the icon to open the program.
Refer to the instructions below to have the 373XL collection program open
at startup.
If any of the data collection folders are not in the default locations, a dialog box
appears asking you to specify where they are located.
The data collection program allows you to indicate preferences for folder
locations, file name conventions, and default settings. To do so, refer to Setting
Preferences on page 5-14.
Note
If communications between the computer and the instrument do not
seem to be working properly, refer to Total Reset of the Instrument on
page 8-8.
To make the data collection program open at startup:
March 2001
1.
Locate and select (single-click) the 373XL data collection program icon.
2.
Choose Make Alias (z-M) from the File menu.
3.
Open the System folder and locate the Startup Items folder.
4.
Drag the 373XL collection alias into the Startup Items folder.
5.
Choose Restart from the Special menu.
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Setting Preferences
The data collection program allows you to indicate preferences for folder
locations, file name conventions, and default settings.
You can choose several types of preferences to set (see Figure 5-3):
•
Folder Locations
•
File Names
For
Sequencing
analysis
•
Sequence Sample Sheet Defaults
•
Sequence Run Defaults
For
GeneScan
analysis
•
GeneScan Sample Sheet Defaults
•
GeneScan Run Defaults
•
General Settings
•
Dye Indicators
The defaults are global parameters that appear in the data entry windows so they
apply to all samples. Since separate Sample Sheets and Run files exist for
sequencing and GeneScan analysis, the defaults you can set are also somewhat
different.
Although the defaults you set appear when you open a new Sample Sheet or Run
file, you can change the fields as required for the individual situation.
Some default settings also appear in the Page Setup dialog box for your particular
printer. They are discussed in the section on printing (see Printing on page 5-67).
Note
5-14
The preferences you set are saved automatically when you close the
Preferences window. You do not need to save each page individually.
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To set preferences:
1.
Choose Preferences from the Window menu and choose the appropriate
option from the submenu that appears.
Figure 5-2. Preferences submenu
2.
Enter or change the defaults, as described on the pages below.
3.
Click OK to accept the changes.
Note
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You can switch from one preferences window to another by using the
pop-up menu labeled “Page:” in the upper left corner of each
preferences window.
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Setting Folder and File Preferences
Setting Folder Location Preferences
The data collection program automatically uses several specific folders for data
storage. You can specify that it use different folders by changing the folder
location preferences.
The Folder Locations page of the preferences dialog box shows the current
folders that store Sample Sheets, modules, runs, the firmware image, and settings
(see Figure 5-3).
To set other preferences select from this pop-up menu. Any
changes you made in this screen are saved automatically
To change the folder where Settings are stored, click this button
Click here to return to the main menu
Figure 5-3. Preferences Folder Locations window
The locations initially shown in this screen are the folders where the indicated
files are normally stored. If you store them in other folders, it is important that you
specify in this screen where you store them. When you create Sample Sheets,
modules, and Run files, the pop-up menus that allow you to choose various
options refer to the folders indicated in this screen.
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•
The Sample Sheet Folder button shows the name and path of the folder in
which you store your Sample Sheets. The Run window pop-up menus that
allow you to choose a Sample Sheet file refer to this folder.
•
The Module Folder button shows the name and path of the folder where
you store modules. This is referred to by the Run window pop-up menus
for plate check, pre-run, and run modules.
•
The Folder containing Run folders button indicates the name and path of
the folder in which Run folders are created when you start a new run.
•
The Firmware Image folder button indicates the name and path of the
folder that contains the Firmware Image file. The first time you turn on the
ABI PRISM 373 DNA Sequencer with XL Upgrade and open the data
collection program, the program looks in this folder for the image file,
which it automatically copies to the instrument.
•
The Settings Folder button indicates the folder containing mobility files,
matrix files, analysis settings files, fragment size standard files, and comb
files. The choices in other Preferences dialog boxes and in the Run
windows refer to this folder for their pop-up menus. The Settings folder is
initially set to be the ABI folder inside your System folder.
IMPORTANT
If you plan to analyze your data using the Sequencing Analysis
software program after data collection, the Settings folder must be set
as the ABI folder.
To change one of the data storage folders:
1.
Choose Preferences from the Window menu and choose Folder Locations
from the submenu that appears.
If you are already in the Preferences window, choose Folder Locations
from the pop-up menu labeled “Page:”.
2.
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Click the button showing the folder's path name to display a dialog box
(Figure 5-4).
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Click here to select
the highlighted folder
Figure 5-4. Folder selection dialog box
3.
Find the new location and click the Select button at the bottom of the file
selection dialog box (Figure 5-4).
IMPORTANT
4.
The data collection software allows you to specify a Settings folder
location. If you analyze your data with the ABI PRISM Sequencing
Analysis program on the computer that you use for data collection, the
Settings folder must always be specified as the ABI folder that is
located in the System folder.
When you have changed all the desired folders, click OK in the Folder
Locations Preferences dialog box (Figure 5-3 on page 5-16) or choose
another preference from the Page pop-up menu.
These folder locations are normally on your local hard disk, but can be on any
AppleShare server volume as well.
Setting File Name Preferences
The data collection program automatically names new files and folders created
during a run (Sample Sheet files, Run folders, Run files, gel files). The Default File
Names page of the preferences dialog shows the method used for automatically
creating file and folder names (see Figure 5-5).
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pop-up menus
allow you to
select suffixes
Enter text in the entry fields to change names
Figure 5-5. Preferences File Names window
A file name can be any text string or nothing at all. By default, the software adds
the date as a suffix, but you can choose to not display a suffix. A Sample file can
have a prefix and a suffix. The prefix can be the lane number or the sample name.
You can choose the suffix from a pop-up menu. The choices are:
•
Nothing <none>
•
Today's date and the current time <date>
•
A serial number reset to one at the start of each run <serial number>
•
A serial number that increments across runs <global serial number>
•
The sample name from the file’s Sample Sheet <sample name>
If you manually save any of these types of files, the name as specified in this dialog
box appears automatically in the Save dialog box. You can change it if you wish.
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To change automatically generated file names:
1.
Choose Preferences from the Window menu and choose File Names from
the submenu that appears.
If you are already in the Preferences window, choose File Names from the
pop-up menu labeled “Page:”.
2.
Enter the desired text in the entry field for each type of
automatically-named file or folder.
The text you enter will be the prefix for all file names of that type.
3.
Specify a suffix by choosing from the pop-up menu to the right of the text
entry field.
4.
When you are finished specifying file name defaults, click OK or choose
another preference from the Page pop-up menu.
Note
5-20
If you choose the “global serial number” option for a suffix, you can
reset the global serial number if necessary. To do so, see page 5-29.
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Setting Defaults for Sequencing Analysis Applications
The following two headings discuss default settings for Sequencing Analysis
applications only. If you are running GeneScan applications, see Setting Defaults for
GeneScan Analysis Applications on page 5-25.
Setting Sequence Sample Sheet Defaults
If you are running sequencing analysis applications, you can set defaults for two
of the choices in the Sample Sheet (Figure 5-6).
The pop-up menu options are files in the
Settings* folder and are filtered for the
correct file type
Figure 5-6. Sequence Sample Sheet Defaults window
Both the DyeSet/Primer and the Instrument file parameters have pop up menus
that allow you to choose none or any dye/set and Instrument file in the Settings*
folder.
To set Sample Sheet defaults for Sequencing Analysis applications:
1.
Choose Preferences from the Window menu and choose Sequence Sample
Sheet Defaults from the submenu that appears.
* If
you are running Sequencing Analysis applications, this folder should be set as the ABI folder in
your System folder (see page 5-17).
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If you are already in the Preferences window, choose Sequence Sample
Sheet Defaults from the pop-up menu labeled “Page:”.
2.
Choose parameters for the two defaults by selecting from the pop-up
menus.
The files listed in the menu are all the applicable files in the Settings folder
(filtered according to type). If you wish to choose a file that is not listed,
choose Other, and a file dialog box allows you to choose any applicable file
in any other folder.
3.
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Click OK or choose another preference from the Page pop-up menu.
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Setting Sequence Run Defaults
You can set several defaults for the Run file of a Sequencing Analysis run (see
Figure 5-7).
To set Run file preferences for Sequencing Analysis applications:
1.
Choose Preferences from the Window menu and choose Sequence Run
Defaults from the submenu that appears.
If you are already in the Preferences window, choose Sequence Run
Defaults from the pop-up menu labeled “Page:”.
Pop-up menus allow you to choose from filtered lists of files. The procedure
below lists where the files are located. To choose a file that is not listed, choose
Other. A file dialog box allows you to choose files from other folders
Figure 5-7. Preferences Sequence Run Defaults window
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2.
Set the defaults as described below.
a.
Enter your name in the Operator entry field.
b.
Specify the separation distance by choosing from the WTR (wellto-read) pop-up menu.
The well- to-read (WTR) distance for sequencing runs is usually 24,
34, or 48 cm.
c.
Click Lanes and choose the number of lanes from the pop-up menu
that appears.
d.
Choose default modules from the PreRun Module and the Run
Module pop-up menus.
The menu shows all Module files in the Module folder (see page
5-17).
e.
To specify that the data be automatically analyzed by the Sequencing
Analysis program, select the checkbox labeled “Autoanalyze with” and
choose Other from the pop-up menu.
In the dialog box that appears, find the analysis program and click
Open.
This specifies the location of the analysis program.
f.
3.
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Select the checkbox at the bottom of the window to specify automatic
printing of the data after analysis.
Click OK or choose another preference from the Page pop-up menu.
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Setting Defaults for GeneScan Analysis Applications
The following three headings discuss default settings for GeneScan Analysis
applications only. If you are running sequencing applications, see Setting Defaults
for Sequencing Analysis Applications on page 5-21.
Setting GeneScan Sample Sheet Defaults
If you are running GeneScan analysis applications, you can set defaults for four
Sample Sheet entries.
To set Sample Sheet defaults for GeneScan analysis applications:
1.
Choose Preferences from the Window menu and choose GeneScan
Sample Sheet Defaults from the submenu that appears.
If you are already in the Preferences window, choose GeneScan Sample
Sheet Defaults from the pop-up menu labeled “Page:”.
Figure 5-8. Preferences GeneScan Sample Sheet Defaults window
• Choose the color for the size standard color you are using.
2.
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Click OK or choose another preference from the Page pop-up menu.
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Setting GeneScan Run Defaults
To set Run file preferences for GeneScan Analysis applications:
1.
Choose Preferences from the Window menu and choose GeneScan Run
Defaults from the submenu that appears.
If you are already in the Preferences window, choose GeneScan Run
Defaults from the pop-up menu labeled “Page:”.
A dialog box appears.
Figure 5-9. Preferences GeneScan Run Defaults window
The default options are the same as for sequencing runs, but the filtered
choices might be different for GeneScan analysis.
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2.
Set the defaults as described below.
a.
Enter your name in the Operator entry field.
b.
Specify the separation distance by choosing from the WTR (wellto-read) pop-up menu.
The well to read (WTR) distance for GeneScan analysis is usually 6,
12, 24, or 34 cm.
c.
Choose the number of lanes from the Lanes pop-up menu.
d.
Choose default modules for the pre-run and the run from the Module
pop-up menus.
The menu shows all Module files in the Modules folder (see page
5-17).
e.
To specify that the data be automatically analyzed by the GeneScan
Analysis program, select the checkbox labeled “Autoanalyze with” and
choose Other from the pop-up menu.
In the dialog box that appears, find the analysis program and click
Open.
This specifies the location of the analysis program.
f.
3.
Select the checkbox at the bottom of the window to specify automatic
printing of the data after analysis.
Click OK or choose another preference from the Page pop-up menu.
General Settings
The General Settings window allows you to specify:
•
A global serial number that increments across runs and can be used in the
names of automatically generated files (see page 5-19).
•
The module to be used for checking the plates.
•
The computer port to which the instrument is attached. Set to modem
port.
The Macintosh computer has two standard connection ports: a Modem
port and a Printer port. Normally, the instrument is attached to the
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computer by means of the Modem port, and a printer or network
connection is attached to the Printer port. If, however, you need to move
the instrument connection to the Printer port, specify that you have made
the change in the general settings. A third option allows you to use the data
collection software when no instrument is attached to the computer.
•
The minimum number of scan lines in a gel.
The size of a gel file is calculated according to the collect time you enter in
the Run window. The number in the General Settings specifies the
minimum number of scan lines for a gel file.
IMPORTANT
Do not change the minimum number of scan lines in a gel. If you make
it smaller, the data collection software might not allocate enough hard
disk space to collect all of your data.
To change the general settings:
1.
Choose Preferences from the Window menu, and then choose General
Settings from the submenu that appears.
If you are already in the Preferences window, choose General Settings from
the pop-up menu labeled “Page:”.
Choose Plate Check
module
Select Modem Port
Keep at 800
Figure 5-10. General settings window
2.
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Make changes as desired.
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• To change the global serial number, select the number in the entry field
and type the new number.
• Choose a plate check module from the pop-up menu.
• If you changed the instrument connection, click the name of the port
to which the instrument is now attached. If you wish to use the data
collection software off-line (not connected to the instrument), click No
Port.
• To change the initial file size of the Gel file, select the number in the
Number of Scans entry field and type a new one. You should not need
to change this setting (see note below).
3.
Note
Click OK or choose another preference from the Page pop-up menu.
The data collection software always performs at least the minimum
number of scans specified in the General Settings window. It performs
more than the minimum specified if necessary to complete a run, based
on the collection time specified in the Run window.
Setting Dye Indicators Defaults
To change the colors that represent the dyes when data is displayed, you can set
Dye Indicators defaults. This is especially useful if you are color blind and the
default settings shipped with the software are not appropriate for your needs.
To set dye indicator preferences for the analysis applications:
1.
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Choose Preferences from the Window menu and choose Dye Indicators
from the submenu that appears.
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If you are already in the Preferences window, choose Dye Indicators from
the pop-up menu labeled “Page:”.
Dye Color is the color that
appears on the computer screen
Plot color is the color that appears
on the printed electropherogram
To change a color, click the button and
choose a new color from the pop-up
Figure 5-11. Preferences Dye Indicators window
2.
Make changes as desired.
• To change a code, type a different character in the appropriate entry
field in the Code column.
• To change a color, click the appropriate color button and choose a new
color from the pop-up menu that appears. The Display Color column
shows the colors used on the computer screen. The Print Color column
shows the colors used for printing.
If you choose Other from the pop-up menu, a color picker appears
(Figure 5-12).
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The upper box shows
the selected color
The lower box shows
the previous color
Click a scrolling arrow
or the color wheel to
change colors
Use the scroll bar to quickly change brightness
Figure 5-12. Color picker
You can choose a new color by using either the scrolling fields to the
left or the color wheel to the right.
To use the scrolling fields, click one of the up or down arrows and
hold down the mouse key. The upper half of the color box shows the
colors as the parameters change. The lower half retains the original
color until you click OK to accept a new color.
To use the color wheel, click the desired color in the wheel. The new
color appears in the upper half of the color box. Move the insertion
point and click again until you find the desired color.
Click OK when you are satisfied with the color.
3.
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When you are finished changing dye indicator preferences, click OK or
choose another preference from the Page pop-up menu.
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Creating and Editing a Sample Sheet
Before starting a run you must record sample information in the Sample Sheet
and the Run file. Once you have set up the Sample Sheet, you can automatically
copy sample information into the Run file, which associates sample information
(name and type of analysis) with each lane position in the gel.
You can choose from two different Sample Sheets (typing or templates),
depending on your application (Sequencing or GeneScan analysis). The only
difference between them is in the entry fields. The entry fields in each are
described under Creating a Sequence Sample Sheet on page 5-34 and Creating a
GeneScan Analysis Sample Sheet on page 5-36. This general information applies to
both.
IMPORTANT
Do not mix sequence analysis and GeneScan analysis samples on the
same Sample Sheet or the same run.
The information you record in the Sample Sheet becomes the sample
identification and other sample-related information in the sample file when the
data is analyzed. The information that you record is critical to the accuracy of
automatic analysis, and is a good reference for you after data collection.
The Sample Sheet is a two-dimensional array, similar to a spreadsheet or ledger.
It consists of rows and columns of fields, each field separated by horizontal and
vertical lines (see Figure 5-13). It accepts more text than fits in the visible fields
and automatically scrolls as you type your entry. To view the beginning of a long
entry, use the keyboard arrow keys. Many of the cells have default values that are
automatically filled in for each sample.
You can use the standard File menu and Edit menu commands in the Sample
Sheet window. You can import from tab-delimited text files (for instance, files
generated by a database) and print the window. You can reuse a Sample Sheet as
often as you wish.
You can export the Sample Sheet as a tab-delimited text file for database,
spreadsheet, or word processing programs (choose Export from the File menu).
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Importing and Exporting Sample Sheet Information
If you wish to import information from a database into the Sample Sheet, first
export the information from the database into a tab-delimited text file. Note the
following:
• The file should contain only the information you wish to import. It should
not have a header.
• The information imported to the first field in the Sample Sheet is
everything up to the first tab in the text file.
• Each row in the text file should contain the information for one row in the
Sample Sheet, in the same order as the Sample Sheet columns.
Exporting a Sample Sheet creates a tab-delimited text file, with the same format
as a file created for import.
To import a text file to the Sample Sheet:
1.
Choose Select All (c-A) from the Edit menu to select all fields in the
Sample Sheet grid.
2.
Choose Import from the File menu.
3.
In the dialog box that appears, choose the file you wish to import.
4.
Click OK.
To export from the Sample Sheet to a text file:
1.
Make sure the Sample Sheet window is active. If it is not, click in it.
2.
Choose Export from the File menu.
3.
In the dialog box that appears, enter a name for the text file, and then
choose where to save the file.
4.
Click OK.
The exported file has an icon like that of a Log file:
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Creating a Sequence Sample Sheet
In the Sequence Sample Sheet you specify dyeset/primer and matrix information
for each sample you plan to run. You can specify default values for both fields by
setting preferences. The defaults appear automatically for each sample, but you
can change them by editing the Sample Sheet. See Setting Defaults for Sequencing
Analysis Applications on page 5-21.
To create a new Sequence Sample Sheet:
1.
Choose New (z- N) from the File menu.
2.
Click the Sequence Sample Sheet icon in the window that appears.
If you set default values they appear in a new Sample Sheet automatically.
Click an arrow to display a pop-up menu of options
Click a title
to select the
entire column
Sample lane
numbers
When you set defaults for mobility and Instrument
files, the columns are filled in automatically
Figure 5-13. The Sequence analysis Sample Sheet
To move from one field to another:
• Click a desired field
• Press the directional arrow keys on the keyboard
• Press Tab to move to the next field to the right
• Press Return to move to the next field down
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3.
Enter/change information as necessary.
Note
To import information from tab-delimited text files, see page 5-33.
a.
In the second column, enter the sample names corresponding to
each lane number.
b.
To choose a DyeSet/Primer mobility file or a multicomponent matrix
other than the default, click the arrow in the field you wish to change
and choose the new file from the pop-up menu that appears.
The DyeSet/Primer files and a default matrix file (the Instrument
file) are provided with your instrument. When you click a field in the
DyeSet/Primer column, the pop-up menu shows only available
DyeSet/Primer files.
The DyeSet/Primer file names indicate information that helps you
choose the correct file. See DyeSet/Primer File Naming Conventions
in your Sequence Analysis User’s Manual.
For most sequencing chemistries the default matrix supplied with
your instrument is appropriate. To read about matrices refer to
Appendix C, Sequencing Instrument File.
Note
To apply the same parameter to all fields in a column, select the
parameter in the top field of the column, click the column title to select
the entire column, and choose Fill Down from the Edit menu.
c.
4.
Note
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To enter a comment, click the comment field. An insertion point
appears, allowing you to type in your comment.
Click the close box to accept the entries.
If you analyze with the wrong dye/primer set, you can use manual
analysis to re-analyze the data against the correct set. Refer to the
ABI PRISM DNA Sequencing Analysis Software User’s Manual.
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Creating a GeneScan Analysis Sample Sheet
In the GeneScan Sample Sheet, in addition to Matrix defaults, you can specify
preferences for the size standard and the dye color used for the standard (see
Setting Defaults for GeneScan Analysis Applications on page 5-25). The defaults appear
automatically for each sample, but you can edit them in the Sample Sheet.
To create a new GeneScan Sample Sheet:
1.
Choose New (z- N) from the File menu.
2.
Click the GeneScan Sample Sheet icon in the window that appears.
Figure 5-14. GeneScan Sample Sheet
To move from one field to another:
• Click a desired field
• Press the directional arrow keys
• Press Tab to move to the next field to the right
• Press Return to move to the next field down
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3.
Enter/change information as necessary.
a.
Type a descriptive sample name in the Sample Name column.
b.
Select a size standard dye color.
The size standard marker (X) appears in the Std field next to the
specified standard dye. You can mark only one size standard for each
sample, and each sample can include up to four dyes per sample.
c.
Enter dye/sample information in the Sample Info column.
The checkbox labeled Used becomes selected so the sample can be
analyzed automatically.
d.
To enter a comment, click the Comments field.
A text entry box appears, allowing you to type in your comments.
Note
To import information from tab-delimited text files, see page 5-33.
IMPORTANT
For downstream analysis and sorting, you must fill out both the Sample
Sheet and the Sample Information window when you create a new
GeneScan Sample Sheet.
4.
Choose Save from the File menu.
Editing a Sequence or GeneScan Analysis Sample Sheet
When you edit Sample Sheets the general procedure is the same, whether it be a
Sequence Sample Sheet or a GeneScan Analysis Sample Sheet. Follow the general
procedure described here, and refer as necessary to the instructions for creating
the individual Sample Sheets.
Note
The Sample Sheet can contain up to 72 rows. If you insert rows beyond
that, the last row in the sheet is dropped off.
You can use the standard Edit menu commands for the File Name, Sample Name,
or Comments fields.
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To modify an existing Sample Sheet:
1.
Choose Open (z- O) from the File menu, and choose the file you want
from the file dialog box that appears.
2.
Edit fields as described for the individual Sample Sheets.
3.
Save the Sample Sheet by choosing Save As from the File menu.
Be sure to save the file under a different name, so you preserve the original
file.
Note
You cannot change a run in progress by editing the Sample Sheet. You
can change the Sample Sheet, but cannot re-select it in the Run
window while the run is in progress. The changes are therefore not
effective for the run in progress.
To move between fields in the Sample Sheet:
• Click a desired field
• Press the directional arrow keys
• Press Tab to move to the next field to the right
• Press Return to move to the next field down
To apply the same parameter to all fields in a column:
1.
Select the parameter in the top field of the column.
2.
Click the column title to select the entire column.
3.
Choose Fill Down (c-D) from the Edit menu.
To copy information from one field to another:
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1.
Click the field to be copied.
2.
Choose Copy (c-C) from the Edit menu.
3.
Click the new field.
4.
Choose Paste (c-V) from the Edit menu.
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To copy an entire row of information to another row:
1.
Click the lane number in the left-most column to select the entire row.
2.
Choose Copy (c-C) from the Edit menu.
3.
Click the lane number of the row you wish to copy into.
4.
Choose Paste (c-V) from the Edit menu.
To move sample information to a different lane:
• Hold down the Option key and click the lane number of the row
containing the information you want to move, and drag the lane number
to another lane number.
When you release the mouse button the sample information appears in the new
location. The lane number does not move.
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Using the Run Window
In the Run window you specify the lane in which each sample will run and the
modules containing the parameters to be used for the run. You also use this
window to start a plate check, a pre-run, or a run.
Separate Run files exist for sequencing and GeneScan analysis applications. Be
sure to choose the correct type for your application.
Opening the Run Window
1.
Choose New from the File menu and click the appropriate Run icon.
This automatically creates a new Run folder, and opens the Run window.
Small document icons open the selected
module or Sample Sheet for review
Specify length of run
Run control
buttons
Pop-up menus in
these panels
allow you to
choose the
modules
(parameters),
the associated
Sample Sheet,
the number of
wells, the
separation
distance (WTR),
and the
Instrument file
for the run
Lane numbers cannot be edited
Select filter set
Figure 5-15. Sequence Run window
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Small document icons open the selected
module or Sample Sheet for review
Specify length of run
Run control
buttons
Pop-up menus in
these panels allow
you to choose the
modules
(parameters), the
associated
Sample Sheet, the
number of wells,
the separation
distance (WTR),
and the matrix for
the run
Lane numbers cannot be edited
Figure 5-16. GeneScan Run window
You need not complete the entire window to perform a plate check. You
must do so to run samples.
Completing a Run Window
You must complete the information in the Run window before pre-running a gel
or running samples.
To complete the Sequence Run window:
1.
Choose the parameters for the run from the pop-up menus.
a.
Choose modules to be used for the plate check, the pre-run and the
run in the Module pop-up menus.
b.
Choose the Sample Sheet from the Sample Sheet pop-up menu.
When you choose the Sample Sheet, the information about the lanes
in which the samples are to run fills in automatically.
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c.
Choose the number of lanes and the separation distance from the
Lanes pop-up menu and the WTR pop-up menu, respectively.
d.
Choose an instrument file from the Instrument File pop-up menu.
Appendix D describes creating and using matrices.
e.
Choose XL Scan or Full Scan in the Run Mode pop-up menu. Use the
table below to determine the appropriate run mode for your lane
configuration.
Lanes
24
32
36
36
48
64
2.
Run Mode
Full Scan
Full Scan
Full Scan
Full Scan
XL Scan
XL Scan
Enter the length of the run in the Collect Time entry field. Following are
some suggested run times for Sequencing runs.
Plate length
(cm)
24
24
34
34
48
3.
Comb
Sharks-tooth
Sharks-tooth
Sharks-tooth
Square-tooth
Sharks-tooth
Sharks-tooth
Type of Run
Full Scan or XL Scan
BaseSprinter
Full Scan or XL Scan
BaseSprinter
Full Scan or XL Scan
Typical Collection Time
(hours)
12
6.5
14
7
16
Enter your name in the Operator entry field.
The sample information for each lane is entered automatically in the grid
at the bottom of the Run window when you choose a Sample Sheet.
Note
4.
5-42
You cannot edit the Sample Sheet information in the Run window.
Make changes in the Sample Sheet, and select the Sample Sheet
again in the Run window to make the changes effective.
Choose the appropriate filter set:
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Applied Biosystems
For Sequenase T7 Terminator chemistry: Select filter set B.
For all other sequencing chemistries: Select filter set A.
5.
Select the Auto Analyze and Auto Print checkboxes if you want the analysis
program to analyze and print the data.
To complete the GeneScan Run window:
1.
Choose the parameters for the run from the pop-up menus.
Note
Any changes you make in your preferences setting will not take effect
until you open a new Run window.
a.
Choose modules to be used for the plate check, the pre-run and the
run in the Module pop-up menus.
b.
Choose the Sample Sheet from the Sample Sheet pop-up menu.
When you choose the Sample Sheet, the information about the lanes
in which the samples are to run fills in automatically.
c.
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Choose the number of lanes and the separation distance from the
Lanes pop-up menu and the WTR pop-up menu, respectively.
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Applied Biosystems
d.
Choose XL Scan or Full Scan in the Run Mode pop-up menu. Use the
table below to determine the appropriate run mode for your lane
configuration.
Lanes
24
24
32
34
36
36
48
50
64
66
Note
Comb
Sharks-tooth
Square-tooth
Sharks-tooth
Square-tooth
Sharks-tooth
Square-tooth
Sharks-tooth
Square-tooth
Sharks-tooth
Square-tooth
Run Mode
Full Scan
Full Scan
Full Scan
Full Scan
Full Scan
Full Scan
XL Scan
XL Scan
XL Scan
XL Scan
If you select a run mode that is incompatible with the selected lane
configuration, the lane popup menu displays <none>.
e.
Choose a matrix file from the Gel Matrix pop-up menu.
Appendix D describes using and creating matrices.
2.
Enter the length of the run in the Collect Time entry field. Following are
some suggested collection times.
Plate length
(cm)
6
12
24
34
3.
Typical Collection Time
(hr)
1.5
4
8
10
Enter your name in the Operator entry field.
As noted, the sample information for each lane is entered automatically in
the grid at the bottom of the Run window when you choose a Sample
Sheet.
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Note
4.
You cannot edit the Sample Sheet information in the Run window.
Make any changes in the Sample Sheet, select <none>, and then
select the Sample Sheet again in the Run window to make the changes
effective.
Choose the appropriate filter set: A or B.
IMPORTANT
Failure to select the appropriate filter set results in the loss of data.
5.
Select the Auto Analyze and Auto Print checkboxes if you want the analysis
program to analyze and print the data.
6.
Complete the information in the Run window, save the file, and start a
plate check or start a pre-run or run, as described in Starting a Pre-run or a
Run on page 5-45.
Starting a Pre-run or a Run
You can start a pre-run or a run either from the Run window or by using menu
commands from the Instrument menu.
IMPORTANT
When starting a run, be sure the sample lanes specified in the Run
window correspond to the lanes you loaded. The sample information in
the Run window is the information stored in the Gel file when you start
the run. Changes you make in the Run window after you start the run
are not implemented.
Starting from the Run Window
When you are finished completing the Run window information, you can start the
run directly from the Run window, as noted above. Instrument control buttons
allow you to start, pause, and cancel the run (see Figure 5-15 on page 5-40).
• Click the PreRun button to pre-run the gel. The button becomes active
when you have entered a module file name for the Pre-run. The Pre-run
lasts for the period of time specified in the pre-run module listed in the
Run window, unless you click the Pause button or the Cancel button.
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Applied Biosystems
Clicking Pre-run causes the instrument to perform electrophoresis, but no
data is collected.
• Click the Run button to start the run. The button becomes active when you
have entered enough information in the window to execute a run. Clicking
Run causes the instrument to perform both electrophoresis and data
collection.
• The Pause button becomes active when you start a run or a pre-run. Click
it to temporarily halt the run (for instance, to load samples). When you do,
the instrument stops electrophoresis. When the run is paused, the Pause
button changes to read Resume. When you are ready to resume the run or
pre-run, click the button again.
Note
If you click Pause during a pre-run to load samples, after all samples
are loaded, click Cancel to stop the pre-run, then click Run to start the
run.
• Click the Cancel button to stop a pre-run or run. If you cancel a pre-run,
the module that is running stops and you can start a run. If you cancel a
run, data collection stops. You cannot continue the run from the point at
which you cancelled it.
Figure 5-17 shows a typical series of commands for a plate check, pre-run, and run
on the ABI PRISM 373XL.
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Button to Click
Module in effect
Plate Check module
n/a
Pre-run module
Task to perform
Check plates and gel for
fluorescence
(Stops plate check scan)
Add buffer and check for leaks
Mount front heat-transfer plate
(Electrophoreses gel without
data collection to equilibrate
temperature)
Prepare samples while
pre-running
Pre-run module
Load first set of samples
during pause
Pre-run module
(Electrophoreses samples into
gel without data collection)
Pre-run module
Load second set of samples
during pause
n/a
Run module
(Stops pre-run)
(Starts electrophoresis and
data collection)
Figure 5-17. Run window buttons used during a typical run
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Starting from the Instrument Menu
The Instrument menu contains commands that perform the same functions as the
Run window buttons (Plate Check, Start PreRun, Start Run, Pause, Resume, and
Cancel Run).
To check the plates for contamination:
Choose Start Plate Check from the Instrument menu.
To start a pre-run:
Choose Start PreRun from the Instrument menu.
To start a run:
Choose Start Run from the Instrument menu.
To temporarily stop a pre-run or run (and resume it later):
Choose Pause from the Instrument menu.
To resume a pre-run or run that has been paused:
Choose Resume from the Instrument menu.
To stop a run before it is finished:
Choose Cancel Run from the Instrument menu. This stops the module that is
running and closes the Run file. You cannot continue the run from the point at
which you cancelled it.
Note
5-48
If you are familiar with running the Model 373 before this
ABI PRISM 373XL DNA Sequencer upgrade, please note the data
collection program no longer has a controller panel; you do not need to
select the Collect button.
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Viewing the Data
Using the Data Collection Program Windows
In the collection program you can open all windows at one time, although the
number might be limited by the available RAM on your Macintosh computer.
Only one window can be active at any given time. Using these windows you can:
• Monitor the instrument status and error occurrence during a run
• Observe the data as it is generated, in the Scan or Gel views, and monitor
the electrophoresis power supply
• Record or change sample information on the Sample Sheet
• Set up and start a run
• Set default settings and parameters for automatic analysis
Viewing the data and the instrument status is described here. Descriptions of the
other functions are found on the following pages:
To perform this function:
See this page:
Record sample information
Page 5-32
Set up and start a run
Page 5-40
Set defaults
Page 5-21 (Sequencing)
Page 5-25 (GeneScan)
Following is some general information about working with windows:
• To move a window around on the screen, click the top border, hold the
mouse button down, and drag the window to another location.
• To change the size of a window, click the size box in the lower right corner
of the window, hold the mouse button down, and drag the mouse to stretch
or shrink the window.
• To close a window, click the close box at the upper left corner of that
window or click in the window to make it active and choose Close (c-W)
from the File menu.
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Observing Instrument Status During and After a Run
During a run, you can observe the current status of the ABI PRISM 373XL in a
status window, which is updated approximately every three seconds. After the run
is finished, you can view a chronological record of significant system events in the
Log file.
The Status Window
Open the Status window by choosing Status from the Window menu.
Click arrows to
collapse /expand
parts of window
Time remaining in
the function being
executed
Green arrow on
scale indicates
actual reading from
the instrument
Gray box to right
and red arrow on
scale indicate
value set for this
parameter by the
chosen module
The total time selected for the
function currently being executed
Figure 5-18. Status Window example
Note
5-50
The actual power and current readings during electrophoresis change
gradually throughout the run. Table 5-5 lists the approximate readings
you should see for power and current under normal running conditions.
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¬
Table 5-5. Readings Under Normal Running Conditions
Gel (%)
6
4.75
4.0
WTR (cm)
24
34
48
Watts
30
32
40
Approximate Readings
(Volts)
Start
End
1200–1500
1800
1200–1500
1800
1500–1700
2100
The Log File
A Run Log file is created for each run and stored in the Run folder when you start
the run. To view the Log file, choose Log from the Window menu.
The Log file contains a comprehensive record of all error and status messages
generated by the data collection program during a sequencing run, including:
• Start and stop times of the run
• Instrument errors and computer errors
These messages can be helpful to Applied Biosystems Field Service Engineers.
Figure 5-19. Log window example
The information in the file is formatted as follows:
xxx
mm/dd/yy hh:mm:ss
description
The entry in the first column, xxx, is variable. The possible entries are:
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...
- ->
<- ***
###
information (system start or stop, file created)
message sent to instrument
message received from instrument
warning
error
The information in the second column shows month/day/year and
hours:minutes:seconds, formatted as shown. The third column has a brief
description of the event.
A Log file can get as big as your available disk space, although you can only view
the last 32K of information using the data collection program. You can use a word
processing program to view larger Log files.
Viewing Data
You can view information from a run in real time during data collection. The Scan
window and the Gel window display the data being collected, and the
Electrophoresis History window displays the set and actual values for the
temperature and the electrophoresis power supply.
The real-time data display differs from the display of data after analysis, as
described next.
Filters and Color of Data Display
On the real-time displays (the Scan window and the Gel window), the data
collection program displays these intensities, color-coded according to
wavelength. Blue, green, yellow, and red (in that order) represent the wavelengths
of the dye emissions within each dye set. Blue represents the shortest wavelength,
and red represents the longest. The colors on the real-time displays therefore
represent the wavelengths of the dyes being detected, rather than the bases being
detected.
Each of the three chemistries used for preparing DNA is associated with one of
the dye sets. Each dye set labels the four bases differently, so the relative
wavelength, and therefore the color, associated with each base varies with the
chemistry used to label it. Because of this, the four colors on the real-time displays
represent different bases, depending on the chemistry used for labeling.
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Table 5-6 describes the colors that represent each of the four bases on the
real-time displays.
Table 5-6. Color Guide for Data Collection Program Raw Data Sequencing Displays
Filter A
Filter B
Color
Taq and T7 Primers
(Dye Set 1)
Taq Terminators
(Dye Set 2)
T7 Terminators
(Dye Set 3)
Blue
C
G
T
Green
A
A
C
Yellow
G
T
A
Red
T
C
G
Note
The Sequencing Analysis program converts the information collected
by the data collection program, so after analysis the colors are the
same for each base: Blue for C, Green for A, Yellow* for G,
and Red for T.
Table 5-7 shows the filter and its band pass wavelength for each filter set, and
which dyes are detected with each filter. For GeneScan applications, it is
important to have a multi-component matrix for every combination of dye sets
you use. In some instances, you may need to make matrices specific for the gel type
as well, as the spectral properties of the dyes are different under denaturing and
native conditions. Because no two band pass filters (of the same wavelength) are
exactly the same, it is necessary to make matrices for each instrument used.
* When
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printed, G is black, so it is easier to see.
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Table 5-7. GeneScan Filter Set Table
Band Pass Filters
Filter Set
A
B
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531
Blue
5-FAM
R-110
6-FAM
545
560
Green
HEX
580
Yellow
TAMRA
Blue
Green
Yellow
Red
5-FAM
6-FAM
TET
HEX
JOE
TAMRA
610
Red
ROX
-R6G
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The Scan Window
Viewing the Scan window is one of three methods of viewing real-time data. Raw
data appears in the Scan window, always in real time. To display the Scan window,
choose Scan from the Window menu.
The Scan display shows the sweeps of the laser across the gel, with a different
colored line representing each filter. The computer simultaneously updates the
Scan window with four lines every few seconds during instrument operation.
Figure 5-20. Scan window example
Note
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You can set the scale of the Scan window by choosing Set Scale from
the Edit menu. A dialog box allows you to enter minimum and maximum
values for the scale.
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The Gel Window
To display the Gel window, choose Gel Image from the Window menu.
Figure 5-21. Gel window example
This window is a reconstruction of actual data similar to the way it would appear
as an autoradiogram, but in color. The first fragments passing the laser appear at
the bottom of the screen and move toward the top of the screen as new data is
recorded throughout the run. By the end of the run, the window appears similar
to an autoradiogram with the smaller DNA fragments near the top and the larger
fragments near the bottom. Remember, however, that this window is a re-creation
of the data through time and does not indicate the physical position of the
fragments.
Note
5-56
You can scroll up or down or resize the Gel window to see the entire gel
image representation.
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The Electrophoresis History Window
To display the electrophoresis history window, choose Electrophoresis History
from the Window menu.
This window displays the set and actual values for the electrophoresis power
supply and gel temperature throughout the course of an electrophoresis run. The
scale for each panel is adjustable (see below).
Click here to adjust scale
Scan numbers
Figure 5-22. Electrophoresis History window example
A green graph represents the actual value; a red graph represents the target.
When the actual and target are identical, the green covers the red. This allows you
to quickly check the status of the instrument visually: red alerts can indicate
potential problems, although some fluctuation is normal.
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Note
The information in the Electrophoresis History window is stored in the
Gel file.
To adjust the scale:
1.
Double-click one of the panels in the window, or click a panel once and
choose Scale from the Edit menu.
Figure 5-23. Set Scale dialog box
2.
In the entry fields of the dialog box that appears, type the minimum and
maximum values you wish to display.
3.
Click OK.
Viewing Other Run-Related Information
The Sample Sheet
To display the Sample Sheet that is associated with the current run, choose
Sample Sheet from the Window menu. This option is available only if a Sample
Sheet is listed in a currently open Run window. See page 5-32 for details about
creating or editing a Sample Sheet.
The Run File
To display the Run file for the current run, choose Run from the Window menu.
See page 5-40 for information about creating the Run file.
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Controlling the ABI PRISM 373 DNA Sequencer
with XL Upgrade Manually
In Manual Control mode, you can change the instrument settings in real time.
Functions you specify from the Manual Control Function pop-up menu are
executed immediately when you click the Execute button, and remain in effect
unless functions in a running module override them. You can run a module
manually only when the instrument is not running any other module.
To control the instrument manually:
1.
Choose Manual Control from the Window menu.
To control individual functions, choose a function from the Fxn pop-up
menu, and enter a value that is within the specified range
Figure 5-24. Manual Control window
2.
Choose a function or a module to run. Table 5-8 on page 5-60 lists the
options.
To choose individual functions:
a.
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Click the Fxn Name field to choose a function from a pop-up menu.
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b.
Enter a value, if necessary, in the Value column of the grid.
The right-most column of the grid lists the range allowed. If you
choose a function that does not take a parameter, No Value appears
in the Value field.
Table 5-8. Manual Control Functions
Function
Range of Values
Electrophoresis Volts*
0.00 to 2.50 kV
Electrophoresis Current*
0.00 to 50.0 mA
Electrophoresis Power*
0 to 300 W
Laser Power
PMT Gain
0.00 to 40.0 mW
(in 10ths of mW)
0 to 999 V
Calibration File Make
no value
Calibration Send Make
no value
* You can manually control these parameters to make any of them limiting,
but you should be thoroughly familiar with the principles of electrophoresis
before doing so.
To choose a module:
a.
3.
Click the Module field to choose the module from a pop-up menu.
Start the chosen function or module.
• To start an individual function: Click the Execute button.
• To start a module: Click the Start button.
To pause or cancel a currently running module, click the corresponding button.
If you click Pause, the text on the button changes to read Resume, and you can
click it again to resume the function or module.
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PMT Gain and Calibration File
Checking and Adjusting the PMT Gain
This section describes, in detail, how to check and adjust the PMT gain. Before
checking or adjusting the PMT gain, you must have
• placed a gel in the instrument, and
• defined the Collection software preferences
Recommended PMT Gain
The PMT gain should be set such that the lowest scan line (usually the blue line)
is positioned at approximately 900 on the y-axis in the Scan window display. For
subsequent runs, the PMT gain needs to be adjusted only if the y-axis value is out
of the range of 800–1000 on the y-axis. Different PMT gains are required for the
three run modes (XL Scan, Full Scan, and BaseSprinter).
Checking the PMT
Use the procedure below to check and adjust the PMT gain for a one-time use or
as part of the procedure to make a calibration file.
To check the PMT gain:
1.
Choose New from the File menu.
2.
Click the Sequence Run icon in the window that appears.
3.
Complete the following information in the Run window.
Note
March 2001
a.
Choose Plate Check from the Plate Check Module pop-up menu.
b.
Choose the appropriate run mode from the Run Mode pop-up menu.
You do not need to complete the other information in the Run window
to perform a plate check or create Calibration File.
4.
Click the Plate Check button.
5.
Allow time for the Translation Processor to update the scan window and
then view the four scan lines in the Scan window.
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6.
Position the mouse so the tip of the arrow just touches a flat section of the
lowest scan line (usually the blue line).
Read y-axis here
Figure 5-25. Scan lines at the recommended range
Note
7.
You can set the scale of the Scan window by choosing Set Scale from
the Edit menu. A dialog box allows you to enter minimum and maximum
values for the scale.
Read the y-axis value displayed in the upper-left hand corner of the scan
window.
• If the lowest scan line is positioned between 800–1000 on the
y-axis, cancel the plate check. Proceed to step 8.
• If the lowest scan line is outside of the recommended range
(800-–1000 on the y-axis), then proceed to Adjusting the PMT Gain.
8.
5-62
Cancel plate check in the run window. If you wish to make a calibration file
for this PMT gain setting, then proceed to Making and Sending a Calibration
File on page 5-63.
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Adjusting the PMT Gain
To adjust the PMT gain:
1.
While the plate check is running, choose Manual Control from the
Window menu.
2.
Adjust the size and position of the Scan and Manual control windows so
that you can see both windows (no overlap).
3.
In the Manual Control window, choose PMT Gain from the Fxn Name
pop-up menu.
4.
Enter a new value then click Execute.
Entering a larger number than displayed in the value box will shift the scan
lines up in the scan window. A smaller number will shift them down.
5.
Click on the scan window to make it active. Allow time for the Translation
Processor to update the scan window , and then check the y-axis value.
6.
Repeat steps 4 and 5 until the PMT gain is within the recommended range.
7.
Cancel the Plate Check. If you wish to make a calibration file for this PMT
gain setting, then proceed to Making and Sending a Calibration File on page
5-63.
Making and Sending a Calibration File
This section describes how to make and send a Calibration file. Before making a
calibration file, you must:
• have a gel loaded in the instrument and
• know how to performed a plate check to obtain a valid PMT gain for each
of the run modes
Calibration File
A calibration file is a single file which stores the PMT gain settings for the three
run modes (XL Scan, Full Scan and BaseSprinter). When a calibration file is
created, the file is named ABI 373XL Calibrations and is stored in the Preferences
folder inside the System folder of the Macintosh. This file can be opened and read
with the SimpleText application.
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Making a Calibration File
The function Calibration File Make in manual control is used store a valid PMT
gain value for each run mode into the ABI 373XL Calibrations file.
To make a calibration file:
1.
Perform a plate check in the XL Scan mode; adjust the PMT gain, if
necessary.
2.
Perform a plate check in the Full Scan mode; adjust the PMT gain, if
necessary.
3.
Perform a plate check in the BaseSprinter mode; adjust the PMT gain, if
necessary.
4.
Choose Manual Control from the Window menu.
5.
Choose Calibration File Make from the Fxn Name pop-up menu.
6.
Click Execute.
7.
The PMT gain values for the three run modes are saved in the ABI 373XL
Calibrations file.
Sending a Calibration File
The function Calibration File Send sends a copy of the information (PMT gains)
in the ABI 373XL Calibrations file to the Translation Processor’s non-volatile
memory. The Translation Processor uses these stored PMT gain values for
subsequent runs.
To send a calibration file:
5-64
1.
Open the Manual Control window, if it is not already open.
2.
Choose Calibration File Send, then click Execute.
3.
A copy of the calibration values now reside in non-volatile memory in the
Translation Processor.
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Saving and Printing Files
Saving Files
The log file created during a run is automatically saved in the corresponding Run
folder. When you close Sample Sheet, run, or preferences windows, the settings in
those windows are stored in the appropriate files. The files are named according
to the default File Names preferences (see Setting File Name Preferences on page
5-18), and are stored in the default folder locations specified in preferences.
You can open most files using the Open command in the File menu, and save
them by choosing Save from the File menu. If you open and change a Sample
Sheet, Run file, or Module window, you might want to save it under a separate file
name to preserve the original information.
To save an altered file under a new name:
1.
Choose Save As… from the File menu.
2.
When the standard file dialog box appears, type a name for your file in the
entry field, and select a location for it.
3.
Click Save.
To save a backup copy of the file you are working with:
1.
Choose Save a Copy In from the File menu.
2.
When the standard file dialog box appears, type a name for your file in the
entry field, and select a location for it.
3.
Click Save.
A backup copy of the file is saved with the new file name, and the original
file remains on your screen.
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Archiving Files
Gel Files
Gel files contain the raw data acquired by the data collection program and
typically take up 10–55MB of disk space. A separate Gel file is created in the Run
folder each time you perform a run. Because Gel files take a substantial amount
of space, you should delete them from your hard disk after you have obtained
satisfactory Sample files. You do not usually need to keep gel files once the
tracking is verified and sample files are created.
If you do wish to archive Gel files, use magnetic tapes, removable cartridge drives,
or optical drives. Gel files are too large to fit on disks.
IMPORTANT
Do not discard any gel file until you have verified the tracking and taken
corrective action if necessary.
Sample files
Use disks, a magnetic tape drive, a removable cartridge drive, or an optical drive
to archive sample files. A 1.4MB, high-density disk holds about six files. A sample
file is 150–200KB in size, depending on the length of the sequencing run. Save a
sample file when you feel confident that the channel selections (tracking) are
correct for the sample.
Printing Files
You can print the contents of any of the editable windows (Sample Sheet, Run
window, Module window).
To specify the location of the printer and the desired printer driver, choose
Chooser from the Apple menu, and then highlight the printer and driver.
Setting Up the Page for Printing
Before you print, you can specify certain parameters about the printout, such as
the paper size, the orientation of the page, and whether or not the print should
be reduced from actual size. To do so, choose Page Setup from the File menu.
The appearance of the Page Setup dialog box depends on the printer you are
using. For information about the other default settings, refer to your printer
manual.
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You must also designate the format of your printed analyzed data through the
appropriate analysis program. Refer to the Sequence Analysis and/or the
GeneScan Analysis preferences.
Printing
Note
If you choose Auto Print for your samples in the Run window,
electropherograms are automatically printed for them after the
collected data is analyzed.
To print the contents of a window:
1.
Make sure the printer is on and loaded with paper.
2.
Click the window you wish to print to make it active.
3.
Choose Print (c-P) or Print One from the File menu.
Print One prints the window immediately, bypassing the standard print
dialog box.
If you choose Print, you can set the number of copies and the pages that
you want to print. The appearance of the print dialog box depends on the
printer you are using.
4.
Click Print.
Importing and Exporting Files
Exporting
Use the Export command to save the contents of a window grid (for instance,
from the Sample Sheet or the Run window) into a tab-delimited text format. This
format is useful with most database, word processing, or spreadsheet programs.
To export information to tab-delimited text format:
1.
Click the window containing the information you wish to export, to make
it active.
The window must be one of the windows containing a grid. The
information in the grid is exported.
2.
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Choose Export from the File menu.
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3.
Type a file name in the dialog box that appears.
4.
Click OK.
Importing
You can also import from tab-delimited text files into the grids of the Sample
Sheet and Run windows. Each row in the text file should contain the information
for one row in the grid, in the same order as the columns of the grid.
To import tab-delimited text files into the data collection program windows:
1.
Open the window into which you wish to import information and click the
top left column of the grid.
1.
Choose Import from the File menu.
2.
In the dialog box that appears, choose the name of the file containing the
text you wish to import.
3.
Click OK.
Note
Everything up to the first tab in the text file is imported into the first field
in the grid.
Quitting the Data Collection Program
Be careful not to quit the data collection program while a run is in progress. If you
do, however, a dialog box appears asking you if you want to quit.
To close any open windows:
Click a window to make it active, then close it in one of the following ways:
•
Click the close box in the upper left corner of the window, or
•
Choose Close from the File menu.
The current settings in the window are saved.
To quit the data collection program:
Choose Quit (z-Q) from the File menu.
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Note
March 2001
If a run is in progress, a dialog box asks you to verify that you want to
quit. Quitting while a run is in progress cancels the run. If data collection
is in progress, the collection is stopped, but collected data is not lost.
You can use either analysis program to open the Gel file containing the
collected data.
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About Data Collection Program Files
Numerous files are stored on the computer that runs the ABI PRISM 373XL. The
application program and various ancillary files are organized in folders on your
hard disk as shown in Figure 5-26 on page 5-71.
The ABI PRISM 373 DNA Sequencer with XL Upgrade folder contains the
program file and folders for Sample Sheets, modules, runs, and settings.
Other files are located in the ABI folder (see Figure 5-26 on page 5-71). This ABI
folder should always be in the System folder on your hard disk. It is the default
Settings folder for the data collection software.
IMPORTANT
5-70
Although you can change the location specified as the Settings folder
in the data collection software, the Settings folder must always be the
ABI folder if you use the Sequencing Analysis program to analyze your
data.
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Folders and Files in the ABI PRISM 373 DNA Sequencer with XL Upgrade Folder
373XL
The Runs folder
contains an
individual folder for
each run. Each
individual run folder
contains the Run file,
the Gel file, the Run
Log, and, after
analysis, the Sample
files
Sample Sheets
you create are
stored in the
Sample Sheets
folder
The Module folder
contains the module
files that provide
instructions to the
instrument during plate
check, pre-run and run
The first time you
start the data
collection program
the software copies
the Image file to the
instrument firmware
Files in the ABI folder in the System Folder
The Settings folder can
contain preferences and
special files used by the data
collection and analysis
programs. The default settings
folder is the ABI folder in the
System folder.
If you use the Sequencing
Analysis software, you must
leave the settings files in the
ABI folder.
Figure 5-26. ABI PRISM 373XL file locations on the computer
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ABI PRISM 373 DNA Sequencer with XL Upgrade Data Flow
Figure 5-27 shows the flow of data between the Macintosh computer and the
ABI PRISM 373 instrument during data collection.
Translation
Interface
Processor
Run instructions
Ra
wd
Run instructions
and
Raw data status
ata
Run File
Sample
Data
, St
Module
files
atu
Sequencing
Analysis
s
Gel File
GeneScan
Analysis
DATA
COLLECTION
PROGRAM
ANALYSIS
PROGRAMS
Electrophoresis
ABI PRISM 373 Instrument
Macintosh Computer
The Run file associates a list of samples with an electrophoresis module. The instrument receives the
run data and sends back raw data to a Gel file, which is passed to the appropriate analysis program.
Figure 5-27. Data collection program data flow
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Input and Output Files
The following describes the files that contribute information for software
operation (input), the files created by the software (output), and the files located
in the ABI folder (which contains both input and output files).
Input Files
The ABI PRISM 373XL input files contribute to operation of the instrument. Table
5-9 details the input files that are not located in the System folder. Output files are
described in Table 5-10 on page 5-74. The files that must be located in the System
folder are described in Table 5-11 on page 5-76.
Table 5-9. Input Files in ABI PRISM 373XL Data Collection Program
March 2001
File Type
Location
Description
ABI PRISM
373 DNA
Sequencer
with XL
Upgrade
Firmware file
(input)
Firmware Image
folder in the
ABI PRISM 373XL
folder
Contains an unalterable copy of firmware code that
the instrument executes in order to run. This file is
copied from the computer to the instrument the first
time you start the ABI PRISM 373XL or if the
instrument memory is cleared after a major power
failure.
Program files ABI PRISM 373XL
folder
Provide the primary input that runs the instrument
and analyzes data. The data collection program
collects and stores the data sent from the
ABI PRISM 373XL during the run.The analysis
program (Sequencing Analysis or GeneScan
Analysis) analyzes this data after the run is
complete.
Run files
Each stores information associating specific
samples with specific lane positions for a particular
run. It also stores information about the Sample
Sheet and modules associated with the run. Files
associated with the run, such as the Run Log, the
Gel file, and the Sample files, are stored in the Run
folder.
Individual Run
folders inside the
Runs folder inside
the
ABI PRISM 373XL
folder
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Table 5-9. Input Files in ABI PRISM 373XL Data Collection Program (continued)
File Type
Location
Description
Sample Sheet Sample Sheets
files
folder inside the
ABI PRISM 373XL
folder
Contain the header information that you entered
into the data collection program Sample Sheet
window. You can export Sample Sheet files in a
format that Microsoft Excel can read.
Module files
Each contains a list of functions that are used by the
instrument during a plate check, a pre-run, or a run.
Module files containing commonly used functions
are provided with the instrument.
Modules folder
inside the
ABI PRISM 373XL
folder
Output Files
The instrument automatically places two output files in the Run folder for an
individual run: a Run file and a status log. Table 5-10 details all of the output files
that are not located in the System folder and their locations. The files that must
be located in the System folder are described in Table 5-11.
Table 5-10. Output Files in ABI PRISM 373XL Data Collection Program
5-74
File type
Location
Description
Gel file
Individual Run
folder inside the
Runs folder inside
the
ABI PRISM 373XL
folder
A large file created by the data collection program.
For a typical run a gel file can be very large (over
20MB). This file contains all raw data acquired by
the data collection program during the run. It is
created in the Run folder when you perform a run.
Run Logs
Individual Run
folder inside the
Runs folder inside
the
ABI PRISM 373XL
folder
Created in the Run folder at the start of each run.
This text file contains date and time-stamped status
and error messages that maintain a comprehensive
history of actions taken, instrument and computer
conditions, and any errors received. You can view
and print the log file at any time while the application
is running.
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Table 5-10. Output Files in ABI PRISM 373XL Data Collection Program (continued)
File type
Location
Description
Sample (data)
files
(Analysis
program
output)
Individual Run
folder inside the
Runs folder inside
the
ABI PRISM 373XL
folder
When you analyze the raw data, the gel file is not
altered. The Sequencing Analysis and GeneScan
Analysis programs* extract sample information from
the gel file to create sample files. Each file contains
the data for a single sample, or lane, including
information you entered in the Sample Sheet; raw
data from the instrument; run start and stop times;
voltage, temperature and power values during the
run; analyzed data; and base calls.
Extracted data depends on tracking: if the tracking
is incorrect, the Sample files are incorrect.
Sequence files
(Analysis
program
output)
Individual Run
folder inside the
Runs folder inside
the
ABI PRISM 373XL
folder
The Sequencing Analysis program also creates
document files called ”.Seq” files (the file names
always end with “.Seq”). These files show the base
letter sequence only (or might contain a header,
depending on the format you choose).You can open
them from word processing programs and print
them. You can also save the files in several formats
that can be read by other software programs. The
Preferences dialog box in the specific analysis
program allows you to choose the file format.
* The Analysis programs are described in detail in separate manuals. Refer to the ABI PRISM Sequencing Analysis
Software User’s Manual or the ABI PRISM GeneScan Analysis Software User’s Manual, as necessary.
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Files Located in the System Folder
The System folder on your Macintosh computer contains both input and output
files. Some are in the ABI subfolder. Others are in the Preferences subfolder.
Table 5-11 lists the ABI PRISM 373XL files located in the System folder.
Table 5-11. Input and Output Files in the System Folder
File type
Subfolder
Description
DyeSet
Primer files*
(input)
ABI folder
Contain dye and primer mobility tables. Applied
Biosystems supplies these files, which are used by
both the data collection and the Sequencing
analysis programs.
Matrix files*
(input)
ABI folder
Contain mathematical matrices that are applied to
data to correct for spectral overlap.
Preferences
file (input)
Preferences folder Maintains information about folder locations and
default file name and settings data.
* Installed by the analysis program
Temporary Files Created in the System Folder
The software programs create a number of temporary files in the System folder. If
you happen to see them, leave them alone. The programs delete them when they
no longer need them.
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6 Instrument Hardware
Contents
About the ABI PRISM 373 DNA Sequencer With XL Upgrade
System Overview
The Loading System
Components
Glass Plates
Spacers
Combs
The Separation System
The Electrophoresis Power Supply
The Buffer Chambers and Electrodes
The Scanning/Detection System
The Read Region and Run Modes
XL Scan Mode
Full Scan Mode
BaseSprinter Mode
Filter Wheel and Data Display
Sequencing
GeneScan
Instrument and Matrix Files
Translation Interface Processor Status and Control
Translation Interface Processor Control
Translation Interface Processor Firmware
ABI PRISM 373 DNA Sequencer with XL Upgrade Data Collection
Software
Status Indicators
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6 Instrument Hardware
3
4
6
6
6
7
7
10
10
10
11
12
12
12
12
13
14
15
15
17
17
18
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About the ABI PRISM 373 DNA Sequencer With XL
Upgrade
The ABI PRISM 373 DNA Sequencer with XL upgrade includes a
microprocessor-controlled electrophoresis and fluorescence detection
instrument, a Power Macintosh computer, and a Translation Interface Processor.
ABI PRISM 373A
Power Macintosh


Macin
Centrtosh
is 650
Translation
Interface
Processor
Figure 6-1. ABI PRISM 373 DNA Sequencer with XL Upgrade
It is an integrated system in which all the components have been carefully
engineered to provide maximum performance.
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The components of the instrument fall into five major categories.
•
Loading:
Gel plates
Spacers
Casting comb
Sharks-tooth comb
Square-tooth comb
Comb overlay
•
Separation:
Electrophoresis power supply
Buffer chambers, electrodes
Heat-transfer plates (Leon and Stretch)
•
Detection:
Argon laser and scanning stage
Filter wheel and PMT
•
Control:
Instrument firmware
Macintosh computer and software.
Main power supply
Translation Interface Processor
System Overview
Using a vertical slab gel, dye-labeled DNA fragments are loaded onto the
instrument. Voltage is applied, causing the fragments to electrophorese through
acrylamide gel and separate according to size.
When the fragments reach a fixed position (the read region) above the lower
buffer chamber, light from the laser excites the dyes, causing them to fluoresce.
The laser beam scans back and forth across the read region of the gel. The emitted
light is collected and focused onto a 5-filter wheel and photomultiplier tube
(PMT). The computer screen displays information received from the Translation
Interface Processor in real time.
At the end of collection, the analysis software can automatically process, analyze,
and translate the collected data into either base sequence, fragment sizing
information, or relative concentrations, depending on your application. The
software also allows you to print the collected data.
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Load DNA
Control
Electrophoresis
Detection


Analysis
Macinto
Centrissh
650
Figure 6-2. ABI PRISM 373 DNA Sequencer with XL upgrade overview
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The Loading System
Components
Glass Plates
Two specially designed glass plates are used to hold the gel through which DNA
electrophoreses.
6, 12, 24 cm WTR
Top
Plain
plate
Notched
plate
16.5 cm
14.5 cm
Read region
16 cm
Plates with spacers and
Figure 6-3. Glass plates and position of read region (Classic and Leon)
6, 12, 24, 48 cm WTR
Top
Notched
plate
34 cm WTR
Plain
plate
Read
region
16 cm
16 cm
10 cm
8 cm
7.5 cm
5.5 cm
Plates with spacers and
Figure 6-4. Glass plates and position of read region (Stretch)
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The region of the glass where the laser scans is called the read region, and is the
located behind the laser beam safety stop when the glass is mounted in the ABI
PRISM 373 DNA Sequencer with XL Upgrade. It occupies an area of approximately
16 cm x 2 cm located in the lower center area of the plates, and its location varies,
depending on the instrument configuration: Classic, Leon, or Stretch (see Figure
6-3 and Figure 6-4 on page 6-6).
Each time you use the glass plates, use them with the same side of each plate on
the inside. After the first use, the notched plate has a hydrophobic area where the
buffer chamber gasket made contact with the plate. You must therefore use the
notched plate in the same orientation each time to avoid difficulty pouring the
gel. It is a good idea to mark the outside of each plate the first time you use it.
Mark the plates with a tiny scratch at the top that doesn’t interfere with the read
region or the upper buffer chamber seal.
Note
Do not touch the cleaned surfaces of the glass plates and wear gloves
to handle the plates.
Spacers
The spacers are small straight plastic pieces that determine the thickness of the
gel. Positioning of the spacers is described in detail in Section 2.
Figure 6-5. Spacers
Combs
ABI PRISM 373 DNA Sequencer with XL Upgrade lane-forming combs are used to
create the individual wells at the top of the gel into which you load your DNA
samples. The Sharks-tooth and square-tooth combs are available for the two
general applications on the ABI PRISM 373 DNA Sequencer with XL Upgrade. A
variety of well numbers is available.
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IMPORTANT
Always use combs, glass plate spacers, and casting combs as a
matched set. Do not mix and match material types or thicknesses within
a set. Mismatching can cause loose-fitting combs, and can result in
sample leaking and/or damage to the gel.
The sharks-tooth comb and casting comb (Figure 6-6) are used with the
Sequencing application of the ABI PRISM 373 DNA Sequencer with XL Upgrade.
The sharks-tooth comb is available with 18, 24, 36, 48, or 64 lanes (wells).
Sharks-tooth comb
Casting comb
Figure 6-6. Sharks-tooth comb and casting comb
After pouring the gel solution, insert the casting comb at the top of the gel to
create a single well or slot. Remove the comb after polymerization.
The sharks-tooth comb is used to form loading wells. Before loading samples,
insert the teeth of the comb into the slot formed by the casting comb during
polymerization. This creates the individual wells into which you load the DNA.
The comb is marked with lane numbers to facilitate loading.
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The square tooth comb (Figure 6-7) is used with the GeneScan application of the
ABI PRISM 373 DNA Sequencer with XL Upgrade. It is available with 34, 36, 50,
and 66 lanes.
Figure 6-7. Square tooth comb
You need to use only the square tooth comb during gel polymerization. After
pouring the gel solution, insert the teeth of the square tooth comb into the
solution so the comb creates individual wells at the top of the gel during gel
polymerization. After polymerization, remove the comb. You do not need to
re-insert it when you load samples.
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The Separation System
The Electrophoresis Power Supply
The electrophoresis power supply provides the electrophoresis field across the
gel. The wattage and voltage are determined by the module you choose in the data
collection software when you start a run. The electrophoresis supply has a
maximum output of 3000 volts, 80 mAmps.
Note
For Sequencing runs and some GeneScan runs, the ABI PRISM 373
DNA Sequencer with XL Upgrade uses constant power, rather than
constant voltage, in order to control temperature.
The Buffer Chambers and Electrodes
The ABI PRISM 373 is supplied with upper and lower buffer chambers.
Color-coded electrophoresis cables, attached to electrodes installed in the
properly filled buffer chambers, attach at the top and bottom of the
electrophoresis chamber, and carry the current that facilitates electrophoresis.
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The Scanning/Detection System
The fluorescent dye labeled fragments passing through the read region (refer to
Figure 6-9 on page 6-12) of the gel are excited by the argon ion laser. The laser
beam is directed into the gel at a constant angle by means of the gel optics, which
are attached to the moving stage. The collection optics are aligned directly in
front of the point at which the laser beam contacts the gel medium. The collected
fluoresced light is then passed in turn through four of the five filters. Each filter
cuts a narrow band of light frequencies corresponding to the emission maxima of
each of the four dyes. Behind the filter wheel is the photomultiplier tube ( PMT).
The signal from the PMT is amplified, integrated, and converted to a digital signal
for transmission to the data acquisition unit (refer to Figure 6-8).
Laser
Mirror
Photomultiplier tube (PMT)
Focusing lens
Filter wheel
Lead screw
Collection lens
Mirror
Focusing lens
Gel
Fluorescent emission
Beam-stop bar
Figure 6-8. Diagram of the laser/scanner assembly (top view)
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The Read Region and Run Modes
The read region is the section of the glass plates and gel where the laser scans
(refer to Figure 6-9). This region is composed of channels at which data is
collected. The ABI PRISM 373 DNA Sequencer with XL Upgrade is equipped with
three data sampling or run modes: XL Scan, Full Scan, and BaseSprinter.
Read region for XL Scan mode:
0–387 channels
Read region for Full Scan mode:
0–193 channels
Read region
Read region for BaseSprinter mode:
0–193 channels
Figure 6-9. The Read region
XL Scan Mode
Data is collected along the entire read region (approximately six inches). The
read region is divided into 388 channels or points where data acquisition occurs.
A single scan of the gel plates with one filter takes 1.5 seconds. A complete scan
with four filters takes six seconds.
Full Scan Mode
Data is collected along the entire read region (approximately six inches) in the
Full Scan mode. The read region is divided into 194 channels or points where data
acquisition occurs. A single scan of the gel plates with one filter takes 1.5 seconds.
A complete scan with four filters takes six seconds. A complete scan equals one
data point or one scan number. Either 194 or 388 channels can be used for the
Full Scan mode.
BaseSprinter Mode
BaseSprinter is available only for Sequencing runs. Data is collected only along the
center section of the read region (approximately three inches). A single scan of
the gel plates with one filter takes 0.75 seconds. A complete scan with four filters
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takes three seconds. A complete scan equals one data point or one scan number.
BaseSprinter mode defaults to 388 channels, although only the middle 194
channels are used.
610 nm filter
580 nm filter
Time
560 nm filter
531 nm filter
0
Channels
193 (or 387)
Figure 6-10. Pictorial representation of sequencing scan mode (filter set A).
Filter Wheel and Data Display
The five bandpass filters contained in the filter wheel are unique to your
instrument. The center bandpasses of the individual filters are 531, 545, 560, 580
and 610 nm. The five filters are split into two sets of four, which are called filter
set A and filter set B. The filters used in filter set A are 531, 560, 580, and 610 nm.
The filters used in filter set B are 531, 545, 560, and 580 nm. The two filter sets
cannot be accessed at the same time.
The filter wheel separates the wavelengths emitted by the fluorescent dyes. On the
real time displays (the Scan and Gel window), the data collection program
displays these intensities, color-coded according to wavelength. Blue, green,
yellow, and red (in that order) represent the wavelengths of the dyes’ emissions
within each dye set. Blue represents the shortest wavelength, and red represents
the longest. Different filter sets use the same four colors to represent different
wavelengths, so the colors do not represent the relative wavelengths of the four
dyes in each dye set.
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Sequencing
Four filters are used for each sequencing run, one for each dye. Filter set A is used
with the fluorescent Dye Primer and Taq Terminator chemistries. Filter set B is
used for the T7 Sequenase Terminator chemistry.
The colors shown for raw data in the Gel and Scan windows (in the data collection
program) reflect the filter used to detect each dye. Since base calling has not
occurred, the colors used do not reflect the bases. Table 6-1 summarizes the
relationship between the filters, dyes, and color display of raw data. In all analyzed
data displays and printed sequencing analysis electropherograms, the colors
correspond to the bases, as follows:
C - Blue, A - Green, G - Yellow (black when printed), T - Red
Table 6-1. Relationship Between Filters, Sequencing Dyes and Data Display
Filter Center
Band (nm)
531
545
560
580
610
6-14
Filter Set A
Taq Primers
Color
Base
Blue
C
--Green
A
Yellow
G
Red
T
Filter Set A
Taq Terminators
Color
Base
Blue
G
--Green
A
Yellow
T
Red
C
6 Instrument Hardware
Filter Set B
T7 Terminators
Color
Base
Blue
T
Green
C
Yellow
A
Red
G
---
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GeneScan
Nine ABI PRISM dyes are available for labeling DNA fragments. The filter set you
choose determines what color peaks appear for a particular dye. Each dye is
detected within its optimum wavelength range through either filter set A or B.
Table 6-2 summarizes the relationship between the filters, dyes, and color display.
Table 6-2. Relationship Between Filters, GeneScan Dyes and Data Display
Filter Set
A
Dyes
B
Color
Display
Filter Center
Band (nm)
Color Filter Center
Display Band (nm)
Dye
HEX
Green
560
Yellow
560
Phosphoramidites
6-FAM
Blue
531
Blue
531
NHS Esters
[F]dNTPs
TET
*
*
Green
545
TAMRA**
Yellow
580
Red
580
ROX
Red
610
*
*
5-FAM***
Blue
531
Blue
531
JOE***
Green
560
Yellow
560
TAMRA
Yellow
580
Red
580
RG6
Green
560
Yellow
560
R110
Blue
531
Blue
531
* Not recommended; the filter is not set at the emission maxima
** TAMRA is available as an NHS-Ester or as an [F]dNTP.
*** 5-FAM and JOE are available only as labeled primers in select reagent kits.
Instrument and Matrix Files
All three sequencing chemistries and GeneScan applications use fluorescent dye
labels that are excited by an argon ion laser. Although the four dyes that are used
fluoresce at different wavelengths, there is some overlapping in the emission
spectra. It is necessary to correct for this overlap (or filter cross-talk) before
analyzing data.
To accomplish this, a mathematical matrix is created for each chemistry type or
dye set, and stored in a special file. For sequencing, this file is called an Instrument
file and contains a separate matrix for each chemistry (dye set) that you run on
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the instrument. For GeneScan, the file is called a matrix file. A different matrix is
required for each combination of the four dyes used, as well as for each type of gel.
When data is analyzed the appropriate matrix is applied to the data to subtract out
any emission overlap. The Instrument file or matrix file is attached to a Gel file or
Sample file when it is created to allow subsequent data analysis. Due to slight
variations in the filters used in the filter wheels, the instrument and matrix files
created on one ABI PRISM 373 DNA Sequencer with XL Upgrade instrument are
not valid on other ABI PRISM 373 DNA Sequencer with XL Upgrade instruments.
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Translation Interface Processor Status and Control
Translation Interface Processor Control
The Translation Interface Processor, which sits on top of the computer’s CPU,
serves as the communication link and data buffer/translator between the ABI
PRISM 373 DNA Sequencer with XL Upgrade and the Power Macintosh.
The power switch is located on the back of the Translation Interface Processor.
Three instrument status lights and a power light are located on the front of the
Translation Interface Processor (see Figure 6-11). The status lights are described
on page 6-19.
The first time you turn on the instrument and start the data collection program,
the program copies a firmware image to the Translation Inteface Processor (refer
to Restarting the Macintosh and Loading the Firmware Image on page 8-10). If you turn
off the instrument between runs, a battery backup holds the image in memory, so
you do not need to copy it again when you restart the system.
Note
March 2001
In order to maintain the optimum charge on the battery, we recommend
you leave the Tranlation Interface Processor on when it is not in use.
6 Instrument Hardware
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Applied Biosystems
Front View
Status LEDs (3)
Power light
Green
Amber
Red
Back View
Status LEDs (9)
Power connector
Power switch
373XL connection
Diagnostics connection
Macintosh connection
Reset button
Figure 6-11. Translation Interface Processor
Translation Interface Processor Firmware
Firmware programmed into the Translation Interface Processor itself controls the
basic functions of the instrument. The Macintosh software communicates
instructions to the firmware, which then operates the hardware.
If the firmware memory becomes corrupted, you might need to clear the
Translation Information Processor’s memory so that the Macintosh copies a new
firmware image into the Translation Interface Processor’s memory the next time
it connects to the Translation Interface Processor. This process is called Resetting
the Firmware Image.
ABI PRISM 373 DNA Sequencer with XL Upgrade Data Collection Software
The ABI PRISM 373 DNA Sequencer with XL Upgrade data collection software
allows you to control and collect data from the instrument. The software
6-18
6 Instrument Hardware
March 2001
Applied Biosystems
communicates instructions to the instrument firmware, which controls the
hardware operation. In addition to displaying data as it is collected, the software
allows you to display instrument status during a run.
After data collection, the data collection software can automatically start the
correct analysis program, if selected.
For more detailed information about the data collection software, refer to
Section 5. For information about the Macintosh computer hardware, refer to
Section 7. Each of the analysis programs has a separate user’s manual.
Status Indicators
Three instrument status lights (LEDs) are located on the front and back of the
Translation Interface Processor. See Figure 8-3 on page 8-9 for the location of the
lights on the back. The LEDs report the status of instrument and indicate if the
instrument’s memory appears to be corrupted.
Three LEDs are located on the front of the instrument (see Figure 6-11 on page
6-18). They indicate instrument status as follows:
March 2001
•
Green indicates that the system is operational or operating. This LED
comes on after completion of the power-on self tests and blinks during a
run unless a special condition causes the amber or red LED to light. At the
end of a run, it stops blinking but remains lit and a blinking amber light
appears with it to indicate that the run is completed and the instrument is
operational. When you open the doors (breaking the interlocks) to
remove the gel, the green light goes steady and the amber light blinks.
When you close the doors again and the system verifies that the interlocks
are engaged, the amber LED goes out and the green LED lights again.
•
Blinking amber indicates that the instrument is operating but paused, or
has lost the firmware image. This LED blinks as the Translation Interface
Processor goes through self-test when you turn the power on. It also blinks
if you open the instrument door (this breaks the laser and electrophoresis
safety interlocks), or if you pause the instrument by selecting Pause from
the Macintosh. At the end of a run, it blinks along with a stable green light
to indicate that the instrument is operational, but is paused because it has
finished the run. A blinking amber light, in the absence of of all others
means the Firmware Image has been lost.
•
Red indicates an error condition that must be corrected for the system to
6 Instrument Hardware
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Applied Biosystems
run properly. This LED lights if an error is detected during self-test. It also
lights if any failures are detected during a run (for example, if the
electrophoresis power or laser fails). Details of errors indicated by the red
LED appear in the log file on the Macintosh. The red LED also lights if
communication between the instrument and the Macintosh fails.
Table 6-3 shows some examples of how the LEDs indicate status during common
and special instrument states.
Table 6-3. System states indicated by status lights
Indicator
State
All off
Instrument off
Green (steady)
Operational
Awaiting Run (no module running)
Passed self-test
Interlocks connected (door closed)
Green (blinking)
Operational
Run in progress
Run resumed after pause
Green (steady)/
Amber (blinking)
End of run
Once instrument door is opened or
a new module is started, the amber
light goes off
Amber (blinking)/
Green (steady)/
Red (steady)
Power-up Self test in
progress
When completed successfully, Red
and Amber goes off and Green
stays lit
Green (blinking)/
Amber (blinking)/
Run paused
Door open
Awaiting Resume
Awaiting door closure
Red (blinking)
Self-test failed
Instrument Failure
Check Macintosh Error Log
Firmware Image lost
Re-load Firmware Image
Amber (blinking)
6-20
6 Instrument Hardware
Notes
March 2001
600 I'M INVISIBLE+6
Applied Biosystems
7 Computer System
Contents
About the ABI PRISM 373 DNA Sequencer with XL Upgrade Computer System 3
System Requirements
3
Installing ABI PRISM 373 DNA Sequencer with XL Upgrade Software
4
Special Software for Data Collection and Analysis
5
What You Need to Know About the Macintosh Computer
6
Maintaining and Caring for Your Macintosh Computer
7
Hard Disk Maintenance
7
Install Only One System Per Hard Disk
8
Back Up All Programs and Files Regularly
8
Restart the Macintosh computer Before Each ABI PRISM 373 DNA
Sequencer with XL Upgrade Run
8
Remove Non-essential Files From the Hard Disk
9
Rebuild the Desktop Regularly
9
Use a Hard Disk Maintenance Program Regularly
9
Use Discretion When Adding Software Programs
10
Error Messages
11
Hard Disk Maintenance Programs
11
Backing Up Important Files Before Optimizing Your Disk
12
March 2001
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About the ABI PRISM 373 DNA Sequencer with XL
Upgrade Computer System
The ABI PRISM 373 DNA Sequencer with XL Upgrade computer system performs
three primary functions:
•
Instrument control
•
Data collection
•
Data analysis
The Macintosh computer provided with your instrument runs special software
(described on page 7-5) to perform these functions.
System Requirements
The ABI PRISM 373 DNA Sequencer with XL Upgrade electrophoresis instrument
is shipped with a Power Macintosh computer. An optional printer is also available.
If you need to replace the computer, or did not order a printer with the
instrument, following are specifications for the type of computer system you
should use with the instrument.
•
Macintosh Model: Power Macintosh (contact an Applied Biosystems
representative for specific models)
• Monitor: Apple Power Macintosh-compatible color monitor, 15-inch display
March 2001
•
Disk Drive: Hard disk with a minimum of 250MB storage (500MB is
recommended)
•
RAM: Minimum 24MB random access memory (32MB is recommended)
Note
About 16MB of RAM should be available at any given time to have both
the data collection and analysis programs open together. To check your
available RAM, choose About This Macintosh… from the Apple ()
menu.
•
Printer: Color printer (contact an Applied Biosystems representative for a
recommendation)
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Applied Biosystems
•
System Software: Macintosh system software version 7.5 or higher
Installing ABI PRISM 373 DNA Sequencer with XL Upgrade
Software
Your field service specialist installs the ABI PRISM 373 DNA Sequencer with XL
Upgrade collection software and analysis software on the Power Macintosh while
installing the instrument.
If you need to re-load the software at some point, this section describes how to
load and configure the programs needed to perform data collection and analysis.
IMPORTANT
Disable the virus protection software on your Macintosh during the
installation process. After installation is complete, you can re-enable
virus protection.
To install the ABI PRISM 373 DNA Sequencer with XL Upgrade software:
Each ABI PRISM 373 DNA Sequencer with XL Upgrade special software program
installed on your computer (data collection and one or two analysis programs, as
described below) is shipped on a separate disk. Follow these steps to install the
ABI PRISM 373 DNA Sequencer with XL Upgrade Data Collection software.
1.
Quit any currently running applications.
2.
Disable any virus protection software and all other inits by holding down
the shift key while choosing Restart from the Special menu.
3.
Insert the Data Collection Install disk into the 3.5-inch disk drive of your
computer.
4.
Click the Installer icon.
5.
Respond to prompts, as appropriate.
The installer script prompts you if you need to insert any other disks, and notifies
you when installation is completed.
Refer to the individual analysis user’s manual for instructions on installing that
application.
7-4
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When you have completed the software installation, we recommend you rebuild
the desktop.
To rebuild the desktop:
1.
Hold down both the command key (z) and Option key and choose
Restart from the Special menu.
2.
Continue to hold down the two keys until the message “Are you sure you
want to rebuild the desktop file...?” appears on the screen.
3.
Click OK.
Special Software for Data Collection and Analysis
The ABI PRISM 373 DNA Sequencer with XL Upgrade comes with software
programs that perform raw data collection and data analysis.
The Data Collection software communicates instructions to firmware on the
instrument that controls instrument function. It also records and displays
instrument status and raw data during a run. You can specify that it automatically
open the analysis program for data analysis after the run is completed.
To analyze the data collected, two other software packages are available,
depending on your application. Normally, one is supplied with your
ABI PRISM 373XL, depending on the configuration you ordered, although you
might have ordered both.
March 2001
•
The Sequencing Analysis software analyzes the raw sequencing data
collected by the data collection program and calls the base sequence. This
program comes bundled with two other programs, GelDocII and
DataUtility. GelDocII allows you to perform special tasks relating to Gel
files. The DataUtility program enables you to make and copy matrices to
correct for the spectral overlap of the fluorescent dye labels. Refer to
Appendix C for instructions on using the DataUtility program. It can also
be used by Applied Biosystems technical specialists to check noise levels.
•
The GeneScan Analysis software enables you to use ABI PRISM 373 DNA
Sequencer with XL Upgrade automated fluorescence detection to size and
quantify DNA fragments. The program automates the entire process of
separating, quantifying, and sizing DNA fragments. It also provides
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Applied Biosystems
flexibility to interactively confirm and fine-tune the data analysis, and
allows you to display the results of an experiment in several different ways.
Each analysis program is described in detail in a separate manual: the ABI PRISM
DNA Sequencing Analysis Software User’s Manual and the ABI PRISM GeneScan Analysis
Software User’s Manual, respectively. For information about the Macintosh
operating system, refer to the Macintosh System 7 Reference manual.
The data collection software and the analysis program(s) you ordered are
installed on the Power Macintosh by your field service specialist when the
instrument is installed, so you do not need to install them yourself.
What You Need to Know About the Macintosh Computer
To run the ABI PRISM 373 DNA Sequencer with XL Upgrade Sequencing System,
you should be familiar with basic Macintosh computer vocabulary and operations,
such as the following:
7-6
•
Using the mouse: clicking and double-clicking, selecting and dragging
•
Choosing commands: using menus, dialog boxes, radio buttons, checkboxes
•
Working with windows: opening and closing, re-sizing and repositioning,
scrolling, understanding the active window
•
Using the Macintosh hierarchical file system: finding files and creating folders
7 Computer System
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Applied Biosystems
Maintaining and Caring for Your Macintosh
Computer
Computers require regular attention and maintenance to operate efficiently and
consistently. Because the ABI PRISM 373 DNA Sequencer with XL Upgrade
software on the Macintosh computer works with large files and accesses the hard
disk often, it is especially important that you follow the procedures described here
to minimize the occurrence of errors during operation. These procedures should
help keep your Macintosh computer operating at maximum efficiency.
The following describes procedures recommended by Apple Computer, Inc. and
Applied Biosystems for regular maintenance of Macintosh computers, and
includes some hints specifically aimed at keeping your ABI PRISM 373 DNA
Sequencer with XL Upgrade software running smoothly.
Hard Disk Maintenance
The following general principles can benefit all Macintosh system users. Follow
these guidelines for optimal performance of the ABI PRISM 373 DNA Sequencer
with XL Upgrade programs and the Macintosh computer.
•
Install only one System per hard disk.
•
Back up all programs and files regularly.
•
Restart the Macintosh computer before each ABI PRISM 373 DNA
Sequencer with XL Upgrade run.
•
Remove non-essential files from the hard disk.
•
Use a hard disk maintenance program every 2–4 weeks (see Use a Hard Disk
Maintenance Program Regularly on page 7-9).
IMPORTANT
•
March 2001
With the large size of gel files and the constant reading and writing to
the hard disk, defragmentation with DiskTools ((included on the Mac
Operating System CD-ROM) or Norton Utilities (from Symantec) is
required every 2 to 4 weeks.
Use discretion when adding software programs, especially extension files.
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Install Only One System Per Hard Disk
The Macintosh computer requires only one System file to operate. This System file
is located in the System Folder on your computer’s hard disk, and is essential for
all operations on the Macintosh computer. The file contains critical information
necessary to monitor and direct all interactions between you and the computer.
Note
Be sure to safely store the System and Utilities disks that came with
your Macintosh computer. Because they are useful if you have
problems starting the hard disk, you should make an extra copy of each
of these important disks.
You do not receive a System file with the ABI PRISM 373 DNA Sequencer with XL
Upgrade programs, but some other applications might furnish an additional
System file. Whenever you copy a program from a disk to your hard disk, avoid
copying additional System files.
For more complete information about the System file and the System Folder, refer
to the Macintosh System 7 Reference manual.
Back Up All Programs and Files Regularly
Although the hard disk is extremely reliable, always back up, or copy onto disks,
the data files on your computer’s hard disk so you do not lose any data. The time
you spend making copies of your files is minimal compared to the time you might
spend if your hard disk fails and you must completely re-create all your files. You
should also keep the original program disks for your software programs in a safe
place, so you can reinstall the programs if necessary.
When you use the ABI PRISM 373 DNA Sequencer with XL Upgrade data
collection and analysis programs regularly, several large data files might
accumulate on your hard disk. These large files can eventually use up all your
available Macintosh storage. Make copies of the data files you do not use regularly
and remove the originals from your hard disk to reclaim storage space for future
work.
Restart the Macintosh computer Before Each ABI PRISM 373 DNA
Sequencer with XL Upgrade Run
Restarting clears the random access memory (RAM) and provides access to the
necessary amount of memory to start data collection.
7-8
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Remove Non-essential Files From the Hard Disk
It is a good idea to remove any unnecessary files from the hard disk before starting
the data collection program. This ensures that you have enough disk space to
store the new Gel file and Sample files for a new run.
To remove a Gel file from your hard disk:
1.
Drag the Gel file into the trash can.
2.
Choose Empty Trash from the Special menu.
The files are not removed from the hard disk until you empty the trash.
Rebuild the Desktop Regularly
Rebuild the desktop monthly and after installing any new software.
To rebuild the desktop:
1.
Hold down the Command (c) and Option keys and choose Restart from
the Special menu.
2.
Continue to hold down the two keys until the message “Are you sure you
want to rebuild the desktop file...?” appears on the screen.
3.
Click OK.
Use a Hard Disk Maintenance Program Regularly
Whenever you write files to the hard disk, and then open and rewrite them, their
physical location on the disk can change. This causes the computer memory to
become “fragmented.” If a significant amount of fragmentation occurs, the system
might run more slowly, and files might be lost. It is extremely important that you
protect your data by running a disk optimizer program regularly.
Optimize the hard disk monthly with Disk Express II, the Speed Disk utility in
Norton Utilities (from Symantec), or a similar program, to prevent the hard disk
from becoming too fragmented. Be sure to back up custom matrix files (in the
folder designated as the Settings folder—refer to Setting Folder Location Preferences
on page 5-16) and any important data before you optimize the hard disk.
March 2001
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Use Discretion When Adding Software Programs
•
Keep SAM Intercept (from Symantec) loaded on your hard disk to check
any disks you insert, and use it to inspect your hard disk either when you
start up or shut down the disk (at least once a day). Make sure you have a
current version of SAM (version 4.08 or later).
Viruses can simply be annoying, but they can also, in the worst case, destroy
all information stored on your hard disk. This could include the System
software, ABI PRISM 373 DNA Sequencer with XL Upgrade software, and
data files.
•
Prevent program conflicts before they occur. Do not load games or other
software programs onto your hard disk. Use the Macintosh computer only
for ABI PRISM 373 DNA Sequencer with XL Upgrade software.
•
Do not use any programs that require the installation of additional
extensions. This includes games that might have custom sounds or
graphics. Use only those that came on your original System Disks, or on the
ABI PRISM 373 DNA Sequencer with XL Upgrade software disks.
Extensions reside in your extension folder in your system folder. They
actually alter the programming code of the system. Compatibility with the
ABI PRISM 373 DNA Sequencer with XL Upgrade software is only
guaranteed for the extensions shipped with your system. Even seemingly
innocuous extensions like After Dark, a screen saver, can interfere with
operations.
While it is unlikely, you might experience conflicts from some extensions
that you think you must use. For example, different printers might have
extensions that access them. If you think a conflict exists, turn off specific
extensions, or all extensions, restart your Macintosh computer, then try
running without the suspect extensions.
You can turn off all extensions by holding down the Shift key while you
restart your computer. You will need to restart it again to turn them back
on.
•
7-10
If your System is ≥ 7.5, you have Extensions Manager. Do not keep the
programs Init Picker, used previously with your Model 373, and Extensions
Manager on the same volume.
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Applied Biosystems
Error Messages
Following the procedures and precautions described above reduces the
probability of errors occurring during ABI PRISM 373 DNA Sequencer with XL
Upgrade runs. Unfortunately, it might not prevent errors from occurring
altogether, so you still might see error messages after performing a run.
Note
If you experience recurring errors, use a hard disk utility program to
check and repair any damage on the hard disk. After it is repaired,
follow all of the preventative maintenance procedures described in this
document.
Hard Disk Maintenance Programs
Following is a limited list of maintenance programs that you can use to check or
maintain your hard disk.
•
Disk Express II
AlSoft
•
Norton Utilities
Symantec
•
Public Utilities
Fifth Generation
•
SilverLining
La Cie
•
CP Optimizer
Central Point Software
•
DiskTools
Included on the Mac Operating System CD-ROM
IMPORTANT
March 2001
Do not use automatic optimization features that might optimize the hard
disk in the background while collecting ABI PRISM 373 DNA Sequencer
with XL Upgrade data. This could cause loss of data.
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Applied Biosystems
Backing Up Important Files Before Optimizing Your Disk
Before optimizing your hard drive, back up all important files onto disks, or
another storage medium, and locate all of the original disks necessary for
reloading the whole system. Proceed as follows:
1.
Back up Instrument and/or matrix files.
a.
Insert a disk into your computer.
b.
Open the System folder.
Note
7-12
If a different folder is designated in Preferences as the Settings folder,
open it instead.
c.
Open the ABI folder.
d.
Drag the Instrument and/or matrix file icons to the disk icon.
2.
Make backup copies of your data.
3.
Optimize your hard drive according to the instructions included with your
software.
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700 I'M INVISIBLE
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8 Maintenance/Troubleshooting
Contents
March 2001
ABI PRISM 373 DNA Sequencer with XL Upgrade Maintenance
Cleaning Instrument Accessories
Replacing Electrophoresis Cables and Electrodes
Upper Buffer Chamber Replacement
Lower Electrode Replacement
Backing Up Important Files Before Optimizing Your Disk
Other Macintosh Computer Maintenance
Troubleshooting
Total Reset of the Instrument
Resetting the Firmware Image and Restarting the Macintosh Computer
Clearing the Firmware Image
Restarting the Macintosh and Loading the Firmware Image
Problems During a Run
Troubleshooting Gels
Software Troubleshooting
Data Collection Run Log Errors
3
3
3
4
6
7
7
8
8
9
9
10
13
14
16
16
8 Maintenance/Troubleshooting
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8 Maintenance/Troubleshooting
March 2001
Applied Biosystems
Maintenance
Most maintenance procedures on the ABI PRISM 373 DNA Sequencer with XL
Upgrade should be performed by a trained Applied Biosystems service technician.
However, you can perform certain procedures yourself, as described below.
Cleaning Instrument Accessories
Be sure to clean the following regularly, as described on the pages noted:
•
Glass plates (pages 2-13 and 3-48)
•
Spacers and combs (page 3-48)
Replacing Electrophoresis Cables and Electrodes
The electrophoresis cables and electrodes are built as assemblies that simplify
installation in the buffer chambers. Change the entire electrode assembly when
you need to replace either a cable or an electrode.
Figure 8-1 shows a drawing of the upper buffer chamber. Figure 8-2 refers to the
lower buffer chamber. Refer to both figures as you perform the following
procedures.
March 2001
8 Maintenance/Troubleshooting
8-3
Applied Biosystems
Upper Buffer Chamber Replacement
Remove the clamps from the old upper buffer chamber and install them onto the
new upper buffer chamber using the following steps (see Figure 8-1).
Right chamber flange
Right clamp
(005018)
Dowel pin
Notch
Black cable
Clamp screw
Left clamp
(005017)
Socket head screw
(212156)
Allen wrench
Figure 8-1. Attach the clamps to the upper buffer chamber
Note
8-4
The buffer chamber should be opaque with the ABI logo stamped on
the lid. If your buffer chamber looks different, contact Applied
Biosystems.
1.
Remove the upper buffer chamber from the instrument and place it on a
work area.
2.
Remove the clamp screw from the right clamp by unwinding the head from
the base of the screw.
3.
Insert the provided Allen wrench through the notch of the right clamp and
loosen the socket head screw by turning it counter-clockwise. If there is no
8 Maintenance/Troubleshooting
March 2001
Applied Biosystems
notch on the clamp, use the shorter end of the Allen wrench to loosen the
socket head screw.
Caution
4.
Remove the right clamp from the flange of the old upper buffer chamber.
5.
Install the right clamp onto the new upper buffer chamber. Make sure that
the dowel pin of the clamp has been inserted into the hole of the right
flange of the new chamber. Use the Allen wrench to tighten the socket
head screw.
6.
Replace the head of the clamp screw on the base.
7.
Repeat steps 2–6 to remove the left clamp from the old upper buffer
chamber
8.
Place the new buffer chamber in the instrument.
9.
Connect the new black cable to the instrument.
WARNING
March 2001
If the clamp cannot be removed because it is attached with
adhesive or the screw is frozen, contact Applied Biosystems for
further instructions.
Use ONLY the opaque upper buffer chamber with the ABI logo on
the lid. If you do have extra upper buffer chambers that are made
of the transparent material do not use them. If you need
replacement chambers contact Applied Biosystems.
8 Maintenance/Troubleshooting
8-5
Applied Biosystems
Lower Electrode Replacement
Replace the electrode of the lower buffer chamber using the following steps (see
Figure 8-2).
Nylon screw 1
(212160)
(do not loosen this screw
Connector
Nylon screw 2
(212179)
Tie holder
(200629)
Nylon washer
(223071)
Feet
Red cable
Electrode
(604175)
Cable clip
(200442)
(do not remove cable
clip from the chamber)
Right support
Figure 8-2. Attach the electrode to the lower buffer chamber
1.
Lift the lower buffer chamber from the instrument and place it on your
work area.
2.
Remove the red cable from the lower buffer chamber and discard.
3.
Partially loosen nylon screw 2 using the screw driver and turn the tie holder
180 degrees.
Nylon screw 2 is located on the inside bottom of the buffer chamber on the
right side when the chamber is facing you.
It is not necessary to loosen or remove nylon screw 1, which is the smaller
nylon screw that attaches the curled cord clip on the left side of the
electrode.
4.
8-6
Remove the old electrode from the lower buffer chamber.
8 Maintenance/Troubleshooting
March 2001
Applied Biosystems
5.
Place the new electrode onto the lower buffer chamber.
The right support of the chamber should be between the electrode’s two
feet.
6.
Insert the cable under the cable clip.
The cable clip stretches around the top of the cable. Rotate the tie holder
to its original position, and use the screw driver to tighten nylon screw 2.
The tie holder and the cable clip should now secure the electrode in place.
7.
Replace the chamber in the instrument.
Plug the new red cable into the instrument. The cable lies on the
instrument between the chamber and the wall of the instrument.
Make a final check that both electrode cables are stored away so that they
won’t interfere with the operation of the instrument door. The new
electrode cables are stronger than the old cables, however, they are also
less flexible so storing the cables may be slightly more difficult.
Note
If in the future either the upper or lower electrode should need to be
replaced, the entire electrode unit will be replaced by ordering a new
electrode from Applied Biosystems. The upper electrode part number
is P/N 604174. The lower electrode part number is P/N 604175.
Backing Up Important Files Before Optimizing Your Disk
Before optimizing your hard drive, back up all important files onto disks or
another storage medium, and locate all of the original disks necessary for
reloading the whole system. Refer to page 7-12.
Other Macintosh Computer Maintenance
Refer to Section 7, Computer System, for routine maintenance issues for the
Macintosh computer.
March 2001
8 Maintenance/Troubleshooting
8-7
Applied Biosystems
Troubleshooting
If you have problems with the instrument, Translation Interface Processor, or the
Macintosh computer, perform a Total Reset. Total Reset consists of several parts:
•
Performing a Total Reset through the keyboard on the instrument
•
Resetting the Firmware Image and restarting the Macintosh computer and
Translation Interface Processor to load the Firmware Image
Total Reset of the Instrument
To perform a total reset of the instrument:
1.
Verify the electrophoresis chamber door is closed.
2.
Press the Main Menu key, and then press the Delete key.
3.
Press Total Reset.
The screen goes blank, then the message, “Calibration in Progress Please
Wait” appears. Total Reset takes about 60 seconds. The instrument is now
ready for use.
8-8
8 Maintenance/Troubleshooting
March 2001
Applied Biosystems
Resetting the Firmware Image and Restarting the Macintosh
Computer
If the firmware memory becomes corrupted, you might need to clear the
instrument’s memory so that the computer copies a new firmware image into
memory the next time it connects to the instrument. Figure 8-3 shows the reset
button and status indicators (LEDs) on the back of the Translation Interface
Processor.
Power switch
Power connector
Status LEDs (9)
373XL connection
Diagnostics connection
Macintosh connection
Reset button
Figure 8-3. ABI PRISM 373XL Translation Interface Procesor (back view)
Clearing the Firmware Image
Possible symptoms of corrupted memory
March 2001
•
The computer does not communicate with the Translation Interface
Processor or the instrument does not appear to run properly (flat or no
scan lines in the Scan window, or no instrument response to computer
commands).
•
The Translation Interface Processor does not communicate with the
instrument or the Macintosh after pressing the reset button, located on the
rear of the Translation Interface Processor.
•
The Translation Interface Processor LEDs (see Figure 8-3 on page 8-9) do
not blink in their characteristic pattern but are stuck in one pattern, which
might be all on or all off AND
•
The Translation Interface Processor might not be able to load a new
Firmware Image on the instrument, OR
8 Maintenance/Troubleshooting
8-9
Applied Biosystems
•
The Translation Interface Processor appears to have loaded a new
Firmware Image onto the instrument but the instrument still does not run
and the Translation Interface Processor LEDs are still not changing.
To clear the Translation Interface Processor’s memory:
1.
Press the reset button on the back of the Translation Processor (refer to
Figure 8-3 on page 8-9.
Use the eraser end of a pencil or something similar.
2.
Watch the back panel LEDs and wait for them to blink in a four-on, four-off
sequence.
In this state, four consecutive lights are on at one time, then all four go off
and the other four go on together.
3.
While the four-on, four-off blink sequence is occurring, press the reset
button a second time within about three seconds after the sequence.
The LEDs change to the “every other one on” state. This indicates that the
Translation Interface Processor’s memory is clear and the Translation
Interface Processor is ready for the computer to copy over a new firmware
image. The next time you start the data collection program, the new
Firmware Image will be copied to the instrument.
Restarting the Macintosh and Loading the Firmware Image
The first time you turn on the ABI PRISM 373 DNA Sequencer with XL Upgrade
and start the data collection program, the program searches for a current version
of firmware on the instrument. If none is located, it automatically copies the
Firmware Image file from the computer to the instrument.
If you turn off the instrument between runs, a battery backup holds the image in
memory, so you do not need to copy it again when you restart the instrument.
Occasionally, however, you might need to copy the image file again, for instance,
if a power outage clears the instrument memory or the memory becomes
corrupted.
To restart the Macintosh to load the Firmware Image:
1.
8-10
Choose Restart from the Special menu in the Finder to restart your
Macintosh.
8 Maintenance/Troubleshooting
March 2001
Applied Biosystems
2.
The data collection software should automatically open upon restarting
the Macintosh. If it does not open, double-click the icon to open the
program. Refer to To make the data collection program open at startup:
on page 5-13 for instructions to open the collection program at startup.
If the program locates the firmware image file it automatically displays a
progress window and starts copying the firmware image file to the
instrument.
When the file is copied, an alert box notifies you to start the data collection
program again.
3.
Click Quit.
4.
Double-click the ABI PRISM 373XL Collection icon to start the program
again.
If, after Step 1, the program does not locate the Firmware Image, a dialog
box appears asking you to identify its location.
March 2001
8 Maintenance/Troubleshooting
8-11
Applied Biosystems
373XL Firmware
373XL Firmware
Figure 8-4. Find Image dialog box
8-12
5.
Locate the ABI PRISM 373XL Firmware file and click Open.
6.
Click Quit when it is finished and restart the ABI PRISM 373XL Collection
program.
Note
If you click Cancel in the Find Image dialog box, the program quits and
returns you to the Macintosh Finder. You cannot start a run without an
ABI PRISM 373XL Firmware Image file.
8 Maintenance/Troubleshooting
March 2001
Applied Biosystems
Problems During a Run
Table 8-1 contains a brief list of problems you might encounter during a run and
what to do about them.
Table 8-1. Problems That Might Occur During Runs
Problem
What to do
Dialog box appears indicating there is not
enough room to store files from run.
Delete files (particularly Gel files) from
the hard disk to make room. Gel files are
stored in each individual Run folder
when you perform a run, and take up a
substantial amount of room on the hard
disk.
Buffer level in upper chamber is getting lower, Siphon the buffer out of the upper buffer
but no external leaks apparent
chamber, dry the corners of the notched
area at the top of the front plate, then
apply a small amount of molten agarose
to the corners of the notches. Refill the
upper buffer chamber. If still l leaking,
you may need to replace the gasket.
March 2001
Buffer splashes and crystallizes around the
read region
Be careful to not overfill the lower buffer
chamber.
Communication problems between the
instrument and the Manintosh
• Check for loose or improperly
connected cables
• Collection software preference set to
No Port. Change preference to Modem
Port.
• Corrupted Firmware. Perform a Total
Reset.
8 Maintenance/Troubleshooting
8-13
Applied Biosystems
Troubleshooting Gels
Table 8-2 shows some problems you might encounter relating to gels, and
recommends some solutions.
Table 8-2. Gel Troubleshooting
8-14
Problem
Possible Cause
Solution
Inconsistent mobilities
from gel to gel
Wrong total percentage
polymer
Follow protocol carefully.
Wrong concentration of Bis
Follow protocol carefully.
Wrong buffer concentration
Follow protocol carefully.
Poor quality reagents
Remake 10X TBE and 40%
acrylamide stock solution
using highest grade
reagents. Urea must also be
ultra pure.
Dissolved O2 concentration
Keep vacuum strength/time
constant; stir/pour gel
solutions gently; filter/pour
gels at 20-23 ˚C.
Variation in spacers
Use spacers and comb sets
that are of equal thickness
Temperature of room, gel
solution or glass varies
20–23 ˚C is optimal
Slow mobility
Old gel
Remake 10X TBE and 40%
acrylamide stock solution
using highest grade
reagents. Urea must also be
ultra pure.
Pour a fresh gel
Poor resolution
Poor quality reagents
(especially acrylamide)
Use fresh reagents from a
reliable source.
Small bubble between load
and read region
Cast gel as described in
protocol.
Well shape not flat
Assure no oxygen trapped
by comb; remove comb
carefully; only load in flat
wells.
8 Maintenance/Troubleshooting
March 2001
Applied Biosystems
Table 8-2. Gel Troubleshooting (continued)
Problem
Catastrophic loss of
resolution (bands tilted
and poorly resolved)
Swirls in gel
Polymerization too slow
March 2001
Possible Cause
Solution
Old buffer
Make 1X TBE fresh daily.
Old gel
Use within 6 hours; do not
refrigerate
Gel extruding from between
plates into upper buffer
chamber
After cleaning plates, wash
briefly in 3M HCl, wash with
water, then clean as
described on page 2-13.
Excessive TEMED or APS
Follow protocol
Temperature too high
Polymerize at 20–23 ˚C
Excessive dissolved O2
Keep vacuum filter strength
and time constant; stir and
pour solutions gently; filter
and pour gels at room
temperature
Not enough TEMED or APS
(or degraded)
Use fresh high-quality
reagents; use as indicated in
protocol
Temperature too low
Polymerize at 20–23 ˚C
Did not use dH2O
Use only distilled or
deionized water for making
all solutions
8 Maintenance/Troubleshooting
8-15
Applied Biosystems
Software Troubleshooting
Data Collection Run Log Errors
If an error message appears on the screen during collection, it also appears in the
Run Log file. The Run Log lists both computer errors and instrument firmware
errors.
Computer errors are individually created and listing each possibility individually
is too massive a task for this manual. Such errors are usually due to problems
related to:
•
Memory
For example, your computer might not have enough memory to handle a
certain function or the data collection program might not have been
allocated enough memory space when it started.
•
File searching
For example, a file might be missing or in a different location so it is not
found when the data collection program tries to open or save it, or a file
might be corrupted.
•
Disk problems
Any problems with your hard disk could cause computer error messages.
Refer to Use a Hard Disk Maintenance Program Regularly on page 7-9.
Check these areas if the Run log lists computer errors.
One instrument firmware error might appear:
•
The AD converter is not functioning.
If you see this message, turn off the power switch, then turn it on again. If the
message appears again, run a plate check to see if a signal is displayed. If a signal
appears, the irregularity is not severe, and you should be able to run samples and
collect data.
8-16
8 Maintenance/Troubleshooting
March 2001
800 I'M INVISIBLE
Applied Biosystems
Appendix A Applied Biosystems Limited Warranty
Applied Biosystems warrants to the Customer that, for a period ending on the
earlier of one year from completion of installation or fifteen (15) months from
the date of shipment to the Customer (the “Warranty Period”), the ABI PRISM 373
DNA Sequencer with XL Upgrade purchased by the Customer (the “Instrument”)
will be free from defects in material and workmanship, and will perform in
accordance with the performance specifications contained in the Instrument
user’s manual that accompanies the instrument (the “Specifications”). During the
Warranty Period, if the Instrument fails to perform in accordance with such
specifications, Applied Biosystems will repair or replace the Instrument at no
charge to the Customer, subject to the conditions below.
This Warranty does not apply to the Instrument's valves, reagent lines, or
performance, unless the Customer uses only reagents and solvents supplied by
Applied Biosystems or expressly recommended by Applied Biosystems, or to
damages caused by reagents or solvents not supplied by Applied Biosystems, even
though recommended by Applied Biosystems. This Warranty does not extend to
any Instrument or part thereof (i) that has been the subjected of misuse, neglect
or accident, (ii) that has been modified or repaired by any party other than
Applied Biosystems or (iii) that has been used in a manner not in accordance with
the instructions contained in the Instrument User's Manual. This Warranty does
not cover the customer-installable consumable parts for the Instrument that are
listed in the Instrument User's Manual.
Applied Biosystems obligation under this Warranty is limited to repairs or
replacements that Applied Biosystems deems necessary to correct covered defects
or failures of which Applied Biosystems is notified prior to expiration of the
Warranty Period. All repairs and replacements under this Warranty shall be
performed by Applied Biosystems on-site at the Customer's location at Applied
Biosystems expense.
No agent, employee, or representative of Applied Biosystems has any authority to
bind Applied Biosystems to any affirmation, representation, or warranty
concerning the Instrument that is not contained in the printed product literature
or this Warranty Statement. Any such affirmation, representation or warranty
made by any agent, employee, or representative of Applied Biosystems shall not
be binding on Applied Biosystems.
Applied Biosystems shall not be liable for any incidental, special, or consequential
loss, damage or expense directly or indirectly arising from the purchase or use of
March 2001
A Applied Biosystems Limited Warranty
A-1
Applied Biosystems
the Instrument. Applied Biosystems makes no warranty whatsoever with regard to
products or parts furnished by third parties; such products or parts will be subject
to the warranties, if any, of their respective manufacturers.
This Warranty is limited to the original Customer and is not transferable.
THIS WARRANTY IS THE SOLE AND EXCLUSIVE WARRANTY AS TO THE
INSTRUMENT AND IS IN LIEU OF ANY OTHER EXPRESS OR IMPLIED
WARRANTIES, INCLUDING, WITHOUT LIMITATION, ANY IMPLIED
WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR
PURPOSE AND OF ANY OTHER OBLIGATION ON THE PART OF APPLIED
BIOSYSTEMS.
A-2
A Applied Biosystems Limited Warranty
March 2001
800 I'M INVISIBLE
Applied Biosystems
Appendix B Quick Reference
Contents
Flow Chart of Typical Sequence Run Process
Flow Chart of Typical GeneScan Run Process
Acrylamide Gel Recipes
Modules
Table of Abbreviations
March 2001
B Quick Reference
3
4
5
6
10
B-1
Applied Biosystems
B-2
B Quick Reference
March 2001
Applied Biosystems
Flow Chart of Typical Sequence Run
Process
Prepare and label samples
Pour gel
Polymerize
~2 hr
Restart Macintosh
Set up software
Load gel on instrument
Run plate check
Cancel plate check
Add buffer
Start pre-run
Pre-run
~5 min
Run samples
into gel
~ 2 min
Resuspend DNA
Pause pre-run
Load first set of samples
Resume pre-run
Pause pre-run
Load second set of samples
Cancel pre-run
Start Run
Run
~ 2–16 hr
Observe status
Analyze and print (automatic)
Clean up
March 2001
B Quick Reference
B-3
Applied Biosystems
Flow Chart of Typical GeneScan Run
Process
Pour gel
Polymerize
~2 hr
Restart Macintosh
Set up software
Load gel on instrument
Run plate check
Cancel plate check
Add buffer,
Start pre-run
Pre-run
~5 min
Run samples
into gel
~ 2 min
Mix sample with loading buffer size std
Heat sample
Cancel Pre-run
Load samples
Start Run
Run
~ 3–8 hr
Observe status
Analyze and print (automatic)
Clean up
B-4
B Quick Reference
March 2001
Applied Biosystems
Acrylamide Gel Recipes
10X TBE
10% Ammonium
Persulfate (APS)
•
•
•
•
• 0.1 g APS
• 1.0 mL dH 2 O
108 g Tris
55 g boric acid
8.3 g EDTA (Na 2 )
Bring to 1 L with
dH2 O
25 mM EDTA 50 mg/mL
Blue Dextran
•
•
•
•
0.93 g EDTA
Add 90 mL dH 2 O
Adjust to pH 8.0
Bring to 100 mL with
dH2 O
• Add 50 mg Blue Dextran
to 1 mL EDTA solution
40% Acrylamide
Stock
• 38 g acrylamide
• 2 g bis-acrylamide•
Bring to 100 mL
with dH2 O
• Deionize
• Filter
4.25% Gel (80 mL)
4.0% Gel (100 mL)
• 10 mL 40%
acrylamide stock
• 50 g urea
• 37 mL dH 2 O
• 1.0 g mixed-bed
resin
•
•
•
•
8.5 mL (40%)
40 g urea
27 mL dH 2 O
1 g resin
• Stir to dissolve
• Filter and degas
• Add 10 mL 10X
TBE
• Bring to 100 mL
with dH2 O
To Polymerize 4.0% Gel
•
•
•
•
Add 500 µL 10% APS
Add 50 µL TEMED
Mix gently
Inject quickly
•
•
•
•
9.5 mL (40%)
40 g urea
27 mL dH 2 O
1 g resin
•
•
•
•
Stir to dissolve
Filter and degas
Add 8 mL 10X TBE
Bring to 80 mL with
dH2 O
6.0% Gel (80 mL)
•
•
•
•
12 mL (40%)
40 g urea
27 mL dH 2 O
1 g resin
To Polymerize 4.25%,
4.75%, and 6% Gels
•
•
•
•
Add 400 µL 10% APS
Add 45 µL TEMED
Mix gently
Inject quickly
To Prepare Sequencing
Sample
• Prepare solution:
• 1 part 25 mM EDTA with
50 mg/mL Blue Dextran
• 5 parts deionized
formamide
•Add to samples
• Mix well
• Heat at 90 °C for 2 min
• Place on ice
B-5
4.75% Gel (80 mL)
To Prepare Matrix
Standards
• Prepare sample:
• 3 µL standard
• 3 µL formamide
• Mix well
• Heat at 90 °C for 2 min
• Place on ice
B Quick Reference
To Prepare GeneScan
Sample
• Add loading cocktail to
sample: Blue EDTA;
formamide, size standard
(see Chapter 4)
• Mix well
• Heat at 95 °C for 5 min
• Place on ice
March 2001
Applied Biosystems
Modules
ABI PRISM 373 DNA Sequencer with XL Upgrade Module
Default Valuesa
Field
(volts)
Power
(watts)b
Current
(mAmp)
Laser
Power
(mW)
Run
Timec
(hr)
--
--
--
40
0.50
PreRun
2500
30
40
40
0.33
Seq Run
2500
30
40
40
12
GS Run
2500
30
40
40
12
Module
Name
Plate Check
a. These default values should be changed according to conditions appropriate to
the gel you are running.
b. These parameters are limited by the Field parameter.
c. Can be set in the Run window
IMPORTANT
March 2001
The Seq Run and GS Run default parameters are applicable only to
24-cm WTR Classic and Leon 6% acrylamide gels.
B Quick Reference
B-6
Applied Biosystems
Throughputs and Filter Sets
ABI PRISM 373 DNA Sequencer with XL Upgrade Throughputs for
Sequencing
Instrument WTR
Gel
Model
Length Concen(cm)
tration
(%)
Scan Mode
Gel
Thickness
(mm)
Power Collec- Length of
(W) tion Time
Read
(hr)
(bases
resolved)
Classic
24
6.0
0.4
XL or Full scan
30
12–14
450
Classic
24
4.75
0.4
BaseSprinter
40
6.5
450
Leon
24
6.0
0.4
XL or Full Scan
30
12–14
450
Leon
24
4.75
0.4
BaseSprinter
40
6.5
450
Stretch
24
6.0
0.4
XL or Full Scan
12–14
450
Stretch
24
4.75
0.4
BaseSprinter
Stretch
34
4.75
0.4
XL or Full Scan
Stretch
34
4.25
0.4
BaseSprinter
Stretch
48
4.0
0.3
XL or Full Scan
261
35
321
45
401
7
450
14–16
550
8
550
17–18
700
1. If necessary, adjust the power to achieve spacing of 9.5 to 12 for analyzed data.
March 2001
B Quick Reference
B-7
Applied Biosystems
ABI PRISM 373 DNA Sequencer with XL Upgrade Throughputs for
GeneScan
B-8
Application
Resolution Size Standard Gel Type
Gel
Run
(bp)
(Acrylamide) Length Time
(cm)
(hr)
Microsatellite Genotyping
(Linkage Mapping Set,
dinucleotide tepeats)
1
GS-350 or
GS-500
6.0%
denaturing
24
6
Microsatellite Genotyping
(tri- and tetranucleotides)
1–2
GS-500
6.0%
denaturing
12
4
VNTRs
1–2
GS-350 or
GS-500
8.0%
denaturing
12
4
Large Fragments
(0.5–2.5 kb)
3–4
GS-1000 or
GS-2500
8.0%
denaturing
6
2
AFLP™ Plant Mapping Kit
1
GS-500
6.0%
denaturing
24
11
Stock Marks™ for Cattle
1
GS-350
6.0%
denaturing
24
6
AmpFlSTR™
(tetranucleotides)
1
GS-500
6.5%
denaturing
24
6
B Quick Reference
March 2001
Applied Biosystems
Dye Sets and Filter Sets by Chemistry
Chemistry
Dye Set
Filter Set
Taq and T7 Primers
1
Filter Set A
Taq Terminators
2
Filter Set A
T7 Terminators
3
Filter Set B
Color Guide for Data Collection Program Raw Data and Gel Image Displays
Filter A
Filter B
Color
Taq and T7 Primers
(Dye Set 1)
Taq Terminators
(Dye Set 2)
T7 Terminators
(Dye Set 3)
Blue
C
G
T
Green
A
A
C
Yellow
G
T
A
Red
T
C
G
Combs and Run Modes
Lanes
B-9
Comb
Run Mode
18
Sharks-tooth
BaseSprinter
24
Sharks-tooth
Full Scan
24
Square-tooth
Full Scan
32
Sharks-tooth
Full Scan
34
Square-tooth
Full Scan
36
Sharks-tooth
Full Scan
36
Square-tooth
Full Scan
48
Sharks-tooth
XLScan
50
Square-tooth
XLScan
64
Sharks-tooth
XLScan
68
Square-tooth
XLScan
B Quick Reference
March 2001
Applied Biosystems
Table of Abbreviations
March 2001
APS
bp
CEHV
COP assay
CsCl
dH2O
Ammonium persulfate
Base pairs
Capillary Electrophoresis High Viscosity
Competitive Oligonucleotide Priming assay
Cesium chloride
Deionized, distilled water
DNA
ddNTP
dNTP
dITP
ds
DTT
EDTA
HCl
IPTG
KH2PO4
Deoxyribonucleic acid
Dideoxynucleoside triphosphate
Deoxynucleoside triphosphate
2'-deoxyinosine-5'- triphosphate
Double stranded
Dithiothreitol
Ethylenediaminetetraacetic acid, -tetracetate, -tetraacetato
Hydrochloric acid
Isopropyl-ß-D-thio-galactopyranoside
Potassium phosphate
kV
mA
min
mM
mL
mRNA
nt
µg
µL
µm
NaCl
NaOH
PCR
PEG
PMT
P/N
q. s.
RF DNA
s
ss
Kilovolts
MilliAmps
Minutes
Milli molar
Milliliter
Messenger RNA
Nucleotide
Microgram
Microliter
Micron or micrometer
Sodium Chloride
Sodium Hydroxide
Polymerase Chain Reaction
Polyethylene glycol 8000
Photomultiplier tube
Part number
Quantity sufficient
Restriction Fragment DNA
Seconds
Single stranded
B Quick Reference
B-10
Applied Biosystems
Table of Abbreviations
STR
SDS
TBE
TE
TEMED
Tris-Cl
VNTR
w
WTR
X-GAL
March 2001
Short tandem repeat
Sodium dodecyl sulfate
Tris-Borate-EDTA (pH 8.3)
10 mM Tris-Cl (pH 8.0), 1 m M EDTA (pH 8.0)
N, N, N', N'-tetramethylethylenediamine
Tris hydrochloride
Variable number of tandem repeats
Watts
Well-to-read or separation length; the distance the sample travels
through the acrylamide before detection, this equals the distance from
the bottom of the well to the laser-reading region.
5-bromo-4-chloro-3-indolyl-ß-galactoside
B Quick Reference
B-11
000 I'M INVISIBLE
Applied Biosystems
Appendix C Sequencing Instrument File
Contents
Sequencing Instrument File
Sequencing Matrix Files in an Instrument File
Filter Wheel and Data Display
Creating a Matrix File
Matrix Standards
Preparing, Loading and Electrophoresing Matrix Standards
Making the First Matrix for a New Instrument File
March 2001
C Sequencing Instrument File
3
3
3
5
5
6
8
C-1
Applied Biosystems
C-2
C Sequencing Instrument File
March 2001
Applied Biosystems
Sequencing Instrument File
Sequencing Matrix Files in an Instrument File
Three chemistries are currently available to prepare DNA samples for sequencing
on the ABI PRISM 373 DNA Sequencer with XL Upgrade: Dye Primers, Taq
DyeDeoxy Terminators, and T7 Terminators. Each chemistry has a specified dye
set: a set of dye labels that emit fluorescence when excited by a laser. Each dye
label in the set emits fluorescence at a different wavelength and, during data
collection, the wavelengths are detected. The wavelengths are separated by
physical filters.
Although the dyes fluoresce at different wavelengths, there is some overlap in the
spectra. To correct for this overlap before analyzing data, a mathematical matrix
is created for each chemistry type (dye set) and stored in a file called the
Instrument file. During data analysis the appropriate matrix is applied to remove
any spectral overlap.
The Instrument file must contain a matrix for each chemistry that you run on the
instrument. When the Gel and Sample files are created, the Instrument file is
attached to each to allow subsequent data analysis. Each ABI PRISM 373 DNA
Sequencer with XL Upgrade must have an Instrument file inside the ABI folder
located in the System Folder.
IMPORTANT
Due to slight variations in the filter wheels, the Instrument file created
on your ABI PRISM 373 DNA Sequencer with XL Upgrade is not valid on
other ABI PRISM 373 DNA Sequencer with XL Upgrade instruments.
Create a new Instrument file only if the filter wheel is replaced, or if you should
need a new matrix for a type of chemistry other than the three mentioned above.
Filter Wheel and Data Display
A filter wheel separates the wavelengths emitted by the fluorescent dyes. On the
real-time displays (the Scan window and the Gel window), the data collection
program displays these intensities, color-coded according to wavelength. Blue,
green, yellow, and red (in that order) represent the wavelengths of the dye
emissions within each dye set. Blue represents the shortest wavelength, and red
March 2001
C Sequencing Instrument File
C-3
Applied Biosystems
represents the longest. The colors on the real-time displays therefore represent
the wavelengths of the dyes being detected, rather than the bases being detected.
Different filter sets use the same four colors to represent different wavelengths, so
the colors do not represent actual wavelengths. They represent the relative
wavelengths of the four dyes in each dye set. Filter A uses the four colors to
represent wavelengths within Dye Set 1 and Dye Set 2. Filter B uses the four colors
to represent wavelengths within Dye Set 3.
Each of the three chemistries used for preparing DNA is associated with one of
the dye sets. Each dye set labels the four bases differently, so the relative
wavelength, and therefore the color, associated with each base varies with the
chemistry used to label it. Because of this, the four colors on the real-time displays
represent different bases, depending on the chemistry used for labeling.
Table C-1 describes the colors that represent each of the four bases on the
real-time displays.
Table D-1. Color guide for data collection program raw data displays
Color
Filter
Band
Pass
Center
(nm)
Filter A
Taq and T7
Primers
(Dye Set 1)
Taq
Terminators
(Dye Set 2)
Filter
Band
Pass
Center
(nm)
Filter B
T7
Terminators
(Dye Set 3)
Blue
531
C
G
531
T
Green
560
A
A
545
C
Yellow
580
G
T
560
A
Red
610
T
C
580
G
The analysis program converts the information collected by the data collection
program, so after analysis the colors are the same for each base. The colors on all
displays of analyzed data, including printed electropherograms, are as follows:
C = Blue
Note
C-4
A = Green
G = Yellow
T = Red
When printed, and when shown against a white background on screen,
G is black so it is easier to see.
C Sequencing Instrument File
March 2001
Applied Biosystems
Creating a Matrix File
Note
If a valid Instrument file exists in the ABI folder (inside the System
folder) on your Macintosh, you need not create one. If you lose your
Instrument file and do not have a backup copy on a floppy disk, refer to
the Sequencing Analysis User’s Manual.
This section describes, in detail, how to make a matrix. The steps you need to
perform are:
1.
Obtain the proper matrix standards for your purpose.
2.
Prepare an acrylamide gel.
3.
Prepare, load and electrophorese the matrix standards.
4.
Refer to the Sequencing Analysis User’s Manual.
Matrix Standards
To make a matrix, you must electrophorese and analyze specific matrix standards.
A separate standard exists for each of the four reactions in each of the three dye
sets. Load only one matrix standard per lane. The matrix standards are sold in kits,
one for each chemistry.
March 2001
C Sequencing Instrument File
C-5
Applied Biosystems
Preparing, Loading and Electrophoresing Matrix Standards
After obtaining the correct matrix standards, follow the procedure below. You can
run matrix standards for Dye Primers and Taq DyeDeoxy terminators on the same
gel, using Filter Set A. Run standards for T7 Terminators on a separate gel using
Filter Set B.
1.
Prepare either a:
• 4.75% acrylamide gel (for Stretch) or a
• 6% acrylamide gel (for Classic and Leon)
ABI PRISM
373XL
Collection
2.
Set up the instrument according to Section 3 of this manual.
3.
Restart the Macintosh (choose Restart from the Special menu in the
Finder).
4.
Start the data collection program.
5.
Verify or define the data collection preferences.
6.
Choose New from the File menu and click the Sequence Sample Sheet
icon.
7.
Fill in the Sample Sheet, as described in Section 3 of this manual.
a.
For convenience, name the matrix standards according to the labeled
base (A, C, G, or T), followed by the specific sequencing chemistry
(Primers, Taq Terminator, or T7 Terminator).
b.
Choose Save As from the File menu to save the Sample Sheet.
Be sure to save it in the folder designated as the Sample Sheets folder
in the data collection program preferences.
8.
Choose New from the File menu and click the Sequence Run icon.
9.
In the Run window, choose the proper files from the pop-up menus.
The Sample Sheet should be the one you completed in Step 7 above.
The Gel Matrix should be the current instrument file or any other
applicable matrix file.
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10. Choose Off from the pop-up menus for Auto Analyze and Auto Print.
a.
Choose Off in the top pop-up menu of the column.
b.
Click the heading of the column to select the entire column.
c.
Choose Fill Down (c-D) from the Edit menu to fill in the other fields.
11. Click the PreRun button to prerun the gel while you are preparing the
standards. Pre-run for 5 minutes, then click Pause in the Run window.
Figure D-1. Completed Sequencing Run window
12. Prepare the Matrix Standards as follows:
March 2001
Note
Do not prepare the matrix standard more than 3 hours in advance of
loading.
Note
As a precaution, prepare and run a duplicate set of matrix standards, in
case one lane is inadequate or the data is lost.
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a.
Briefly vortex the matrix standards to ensure the solutions are well
mixed. Spin the solution briefly to collect the fluid at the bottom of
the tube.
b.
For each standard (one tube for each base), combine 3 µL of standard
with 3 µL of deionized formamide.
c.
Briefly vortex each of the tubes prepared above to ensure that the
sample is well mixed. Spin the solution briefly to collect the fluid at
the bottom of the tube.
d.
Heat all matrix samples at 90˚C for 2 minutes. Immediately place
samples on ice until loaded.
13. Load the matrix standards in the lanes you designated in the Sample Sheet.
Comb Configuration
Loading Volume (µL)
18
24
32/36
48
64
4
4–6
4
1.5–2
1–1.5
14. Click Run to start the run.
A dialog box appears, allowing you to change the name and location of the
gel file created by the run.
15. Proceed to either Making the First Matrix for a New Instrument File, or Adding
or Replacing a Matrix to an Instrument File after the sequencing run is
complete.
Making the First Matrix for a New Instrument File
If you need to re-create a matrix file for any reason, you should:
• Run the appropriate matrix standards
• Check the gel file to verify that lane tracking is correct and that peaks exist
in the raw data.
• Make the matrix file, using the DataUtility program.
• Verify the accuracy of the matrix file, and its location.
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Follow these steps (usually along with your Service Representative) when you get
a new instrument.
Note
If you lose your Instrument file and do not have a backup copy on a
floppy disk refer to the Sequencing Analysis User’s Manual before
re-creating the entire Instrument file.
Now that the matrix standards have been electrophoresed, switch to the matrix
making instructions in the Sequencing Analysis User’s Manual.
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Appendix D GeneScan Matrices
Contents
Introduction
Filter Wheel and Data Display
Creating a Matrix File
Matrix Standards
Preparing, Loading and Electrophoresing Matrix Standards
Creating the Matrix File
Applying and Verifying the Matrix
Method 1
Method 2
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3
4
6
6
6
10
13
13
15
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Introduction
Although the dyes used to label your fragments fluoresce at different wavelengths,
there is some overlap in the spectra. To correct for this overlap before analyzing
data, the software uses a matrix file, which contains a mathematical matrix.
You create one matrix for each chemistry and gel type. During data analysis the
appropriate matrix is applied to remove any spectral overlap.
A specifically created matrix file provides the best results for your particular
instrument and the dye set and run conditions used in the experiment. In most
cases, you should not need to modify the matrix file once you have created it.
You may want to create a new matrix to accommodate these conditions:
•
If you use gel materials or buffers with pH values that differ greatly from
one another (in such a case you need different matrices)
•
If you change dyes in a dye set, e.g., changing from 5-FAM to 6-FAM. Even
though the dyes represent the same color, their spectral characteristics are
different.
•
If you use dyes other than those provided by Applied Biosystems
•
If you see problems that appear to be the result of spectral overlap
Once you create a matrix file, you can apply that file to any run made under the
same conditions, using the same dye set, and on the same instrument.
Applied Biosystemss provides Matrix Standard kits that are designed to test and
define the multicomponent matrices for specific dye sets. To work with a matrix
file, use the Matrix Standard kit that was designed for the specific dye set you plan
to use. Run the matrix standards of different types in different lanes to make the
matrix and to test whether the spectral overlap of the dyes is corrected by the
matrix.
IMPORTANT
March 2001
To specify matrix file in the data collection program so you can perform
automatic analysis, you must store the matrix files in the ABI folder
inside your System folder.
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Filter Wheel and Data Display
The band pass filters on the filter wheel separate the wavelengths emitted by the
fluorescent dyes. On the real-time displays (the Scan window and the Gel
window), the data collection program displays these intensities, color-coded
according to wavelength. Blue, green, yellow, and red (in that order) represent
the wavelengths of the dye emissions within each dye set. Blue represents the
shortest wavelength, and red represents the longest. The colors on the real-time
displays therefore represent the wavelengths of the dyes being detected, rather
than the fragments being detected.
Different filter sets use the same four colors to represent different wavelengths, so
the colors do not represent actual wavelengths. They represent the relative
wavelengths of the four dyes in each dye set.
The filter set you choose determines what color peaks appear for a particular dye
(see Table D-1). Through either filter set A or B, each dye is detected within its
optimum wavelength range. It is important to have a matrix for every combination
of dye sets you use.
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Table D-1. ABI PRISM dyes and filter set color displays
Filter Set
A
Dyes
B
Color
Display
Filter Center
Band (nm)
Color Filter Center
Display Band (nm)
Dye
HEX
Green
560
phosphoramidites
6-FAM
Blue
531
Blue
531
TET
*
*
Green
545
TAMRA**
Yellow
580
Red
580
ROX
Red
610
*
*
NHS Esters
[F]dNTPs
Yellow
560
5-FAM***
Blue
531
Blue
531
JOE***
Green
560
Yellow
560
TAMRA
Yellow
580
Red
580
RG6
Green
560
Yellow
560
R110
Blue
531
Blue
531
* Not recommended; the filter is not set at the emission maxima
** TAMRA is available as an NHS-Ester or as an [F]dNTP.
*** 5-FAM and JOE are available only as labeled primers in select reagent kits.
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Creating a Matrix File
To make a matrix:
1.
Obtain the appropriate matrix standards.
2.
Prepare an acrylamide gel.
3.
Prepare, load, and electrophorese the matrix standards.
4.
Follow the procedure on page D-6.
Matrix Standards
To make a matrix, you must electrophorese and analyze specific matrix standards.
Separate standards exist for each dye. Each dye used must be recognized as a
different color by a single filter (Table D-1 on page D -5). Table D-2 lists the kits
and their part numbers.
Table D-2. Matrix Standard Dyes and Part Numbers
Matrix Standards
Part Number
Dye Primer (5-FAM, JOE, TAMRA, ROX)
401114
Fluorescent Amidite (6-FAM, HEX, TET, TAMRA, ROX)
401456
Preparing, Loading and Electrophoresing Matrix Standards
To set up the instrument:
1.
Prepare the appropriate gel.
2.
Set up the instrument according to Section 4 of this manual.
3.
Restart the computer (choose Restart from the Special menu in the
Finder).
To set up the GeneScan Analysis software:
1.
Start the GeneScan Analysis program.
2.
Define the gel processing parameters.
a.
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Choose Gel Processing Parameters from the Settings menu.
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3.
4.
b.
Select both boxes for Auto-Launch Processing.
c.
Set the Scan Range from 0 to 10000.
d.
Deselect Multicomponent.
e.
Click OK.
Define the analysis parameters.
a.
Choose Analysis Parameters from the Settings menu.
b.
Define all the appropriate parameters.
c.
Select Baseline and deselect Multicomponent boxes.
d.
Click OK.
Define the project options.
a.
Choose Project Options from the Settings menu.
b.
Complete the each of the submenus.
To complete the set up:
1.
Start the data collection program.
2.
Choose Preferences from the Window menu to verify or set the data
collection preferences.
3.
Choose New from the File menu, and then click the GeneScan Sample
icon.
4.
Fill in the Sample Sheet as described in Section 4 of this manual.
a.
In the sample name column, type in a name appropriate to the matrix
standard loaded.
b.
Choose Save As from the File menu to save the Sample Sheet.
Be sure to save it in the folder designated as the Sample Sheets folder
in the data collection program preferences.
5.
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Choose New from the File menu, and then click the GeneScan Run icon.
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6.
In the Run window, choose the proper files from the pop-up menus.
The Sample Sheet should be the one you completed in Step 15, above.
The Gel Matrix should be <none>.
7.
8.
9.
Choose Off from the pop-up menus for Auto Analyze and Auto Print.
a.
Choose Off in the top pop-up menu of the column.
b.
Click the heading of the column to select the entire column.
c.
Choose Fill Down (c-D) from the Edit menu to fill in the other fields.
Click Plate Check.
a.
Check the plates for extra peaks. Reclean, if necessary.
b.
Set or verify the PMT setting.
Click PreRun to prerun the gel while you are preparing the standards. Prerun for 5 minutes, and then click Cancel in the Run window.
Figure D-1Completed GeneScan Run window
10. Prepare the Matrix Standards as follows:
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Note
Do not prepare the matrix standard more than 3 hours in advance of
loading.
Note
As a precaution, prepare and run a duplicate set of matrix standards, in
case one lane is inadequate or the data is lost.
a.
Briefly vortex the matrix standards to ensure the solutions are well
mixed. Spin the solution briefly to collect the fluid at the bottom of
the tube.
b.
For each standard (one tube for each base), combine 1 part standard
with 1 part deionized formamide.
c.
Briefly vortex each of the tubes prepared above to ensure that the
sample is well mixed. Spin the solution briefly to collect the fluid at
the bottom of the tube.
d.
Heat all matrix samples at 90˚C for 2 minutes. Immediately place
samples on ice until loaded.
11. Flush the wells with loading buffer.
12. Load the matrix standards in the lanes you designated in the Sample Sheet.
Use the following table to determine the load volumes for your matrix run.
Number of Wells Load Volume (µL)
24
5
34/36
4
50
1.5–2
66
1–1.5
13. Click Run to start the run.
A dialog box appears, allowing you to change the name and location of the
gel file created by the run. Click Save.
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Creating the Matrix File
After preparing, loading, and electrophoresing matrix standards, you verify lane
tracking and peaks in the raw data, and then create the matrix.
To verify lane tracking and peaks in the raw data:
1.
Start the GeneScan Analysis program.
2.
Choose Open Gel from the File menu and open the gel containing the
matrix standards.
3.
Check the Gel File window to see if the lane tracking was successful for
each standard, and correct any gel tracking problems.
Note
If you are unfamiliar with how to retrack a gel file and view data, refer to
the ABI PRISM DNA GeneScan Analysis User’s Manual for detailed
instructions.
To create the matrix and print the values:
1.
Determine the range of scan points to be used for making the matrix.
a.
Click the name of the four matrix standard sample files in the Analysis
Control window and choose Raw Data (z-R) from the Project menu.
b.
Select a starting data point that starts at a flat baseline in the middle
of the run, well after the primer peaks. The range needs to contain a
minimum of three well-defined peaks or 1000 data points.
Typically, the same start point and range is used on all four matrix
standard sample files.
c.
2.
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Close the raw data windows.
Choose New from the File menu, and then click the Matrix icon in the
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window that appears.
Figure D-2. Make New Matrix dialog box
March 2001
a.
Click the B… button.
b.
In the file dialog box that appears, choose the Sample file containing
your blue dye matrix standard.
c.
Enter the Start At point as determined in step 1.
d.
Repeat steps a through c for the other three dyes.
e.
Enter the number of data points to include, at least three well-defined
peaks and 1000 points.
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f.
Click OK.
The Matrix Values window appears, showing the values used to calculate
the overlap correction.
Figure D-3. Matrix window example
For each dye, the value where the dye fluorescence is read by the
appropriate filter is 1.000. The adjacent colors show the amount of overlap
for which the system needs to compensate.
3.
Choose Save from the File menu, and give the new matrix file a unique,
descriptive name.
Note
Save all matrix files in the ABI folder in the System folder and name
matrix files by instrument serial number and type of gel used, for
example, “#273 TBE den urea.”
It is also helpful to include “GS” in the name of the matrix files to
differentiate between sequencing and GeneScan applications.
4.
Choose Print (c-P) or Print One from the File menu to print the matrix
values.
You can use this printout for documentation.
5.
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Click the Close box to close the window.
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Applying and Verifying the Matrix
To apply and verify the matrix:
Use either of the following two methods to check the quality of the generated
matrix.
Method 1
1.
Apply the new matrix to your matrix sample files within a project.
a.
Choose Analysis Control from the Windows menu.
b.
Select the matrix standard sample files.
Shift-click to select multiple consecutive files or c-click to select
multiple files that are not consecutive.
2.
c.
Choose Assign New Matrix from the Project menu.
d.
In the directory box that appears, find and select the newly created
matrix.
e.
Click Open.
Verify the matrix.
a.
Choose Analysis Parameters from the Settings menu.
b.
Select Baseline and Multicomponent (and any other applicable
parameters).
c.
Select the matrix sample files and the colors to be analyzed (B, G, Y,
and R buttons).
d.
Click Analyze.
e.
Choose Results Control from the Windows menu.
f.
Display the analyzed data.
For each sample file, the only visible peaks should represent the dye
loaded in that lane. All other lines should be relatively flat. This
indicates that the matrix properly compensated for the spectral
overlap.
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A matrix that does not compensate for the other dyes (see Figure D-1) is
unacceptable and must be regenerated. If necessary, the gel and matrix standards
must be rerun. Examples of good and poor matrices applied to the same 5-FAM
data (12-cm run) are shown in Figure D-1 and Figure D-1, respectively.
Figure D-1Good matrix—only blue peaks are present
Figure D-1Poor matrix—note the other peaks at the same scan number as the blue
peaks
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Method 2
1.
2.
a.
With the collection gel open, choose Attach New Gel Matrix from the
Gel menu.
b.
Find and select the newly created matrix in the dialog box that
appears.
c.
Click Open.
Define the gel processing parameters.
a.
Choose Gel Processing Parameters from the Settings menu.
b.
Select the appropriate Scan range (excluding excess primer peak).
c.
Select Multicomponent and baseline.
3.
Choose Regenerate Gel Images from the Gel menu.
4.
Use the “slice view” to examine the multicomponented sample files.
5.
March 2001
Apply the new matrix to your gel file.
a.
On the gel display, click the triangle above the lane which represents
one of the matrix standards.
b.
To the left of the gel image is a slice view. Use this view to examine the
data.
c.
Verify that the only visible peaks correspond to dye-labeled fragments
loaded in the lane. All other lines should be relatively flat. This
indicates that the matrix properly compensated for the spectral
overlap.
d.
Repeat steps a through c for the remaining sample files.
Apply the new matrix to the sample files within a project. Refer to
page D-4 and page D-5 for help interpreting data.
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Appendix E Optimizing PCR
Contents
Preparing Reaction Mixtures
Reaction Volume and Tube Types
Deoxynucleoside Triphosphate (dNTP) Concentration
Magnesium Ion Concentration
Primer Concentration
Template Concentration
Enzyme Concentration
Performing Hot Start PCR
Setting Temperature Control Parameters
Avoiding Contamination
Preventing PCR Product Carryover
Cleaning Up Spills on Work Surfaces
Analyzing Post-PCR Products
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3
5
5
5
6
6
8
10
12
12
13
14
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Preparing Reaction Mixtures
A reaction mixture consists of all the components you put into a reaction tube
before loading the tubes in a thermal cycler and running a PCR reaction. When
preparing reaction mixtures, consider the following factors that can affect overall
yield of specific DNA target sequences:
•
Reaction volume and tube types
•
Deoxynucleoside triphosphate (dNTP) concentration
•
Magnesium ion concentration
•
Primer concentration
•
Template concentration
•
Enzyme concentration
Reaction Volume and Tube Types
When using an Applied Biosystems PCR instrument system, reaction volumes may
cover the range of 5–100 µL, but should not exceed 100 µL. If you use volumes
larger than100 µL, you may need to program longer incubation times to assure
adequate thermal equilibration of the reaction mixture.
Table E-1 on page E -4 shows the types of reaction tubes that you can use with
each PCR instrument system.
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Table E-1. Reaction tube types for PCR instrument systems
PCR Instrument
System
Reaction Tube Type
Uses
DNA Thermal Cycler
GeneAmp® PCR
Reaction Tubes
Optimized for fast PCR
amplification of reaction
volumes between 25 µL
and 100 µL.
DNA Thermal Cycler
480
0.5 mL GeneAmp
Thin-Walled Reaction
Tubesa
Allows you to program
shorter hold times
(45 sec. or more) at each
temperature in the PCR
cycle.
GeneAmp PCR
System 9600
0.2 mL Thin-Walled
MicroAmp® Reaction
Tubes
Optimized for fast PCR
amplification of reaction
volumes between 5 µL
and 100 µL.
Can be used without
mineral oil.
0.5 mL Thin-Walled
MicroAmp Reaction
Tubes
Optimized for fast PCR
amplification of 100 µL
reaction volumes.
Use with either an oil
overlay or AmpliWax
PCR Gem beads.
0.2 mL MicroAmp
Reaction Tubes
Optimized for fast PCR
amplification of reaction
volumes between 5 µL
and 100 µL.
GeneAmp PCR
System 2400
a. Can also be used with the DNA Thermal Cycler if you set hold times of at least 60 seconds.
Although reaction tubes usually do not need to be sterilized or siliconized, you
should autoclave tubes when dealing with small quantities of starting DNA
template.
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Deoxynucleoside Triphosphate (dNTP) Concentration
In the standard GeneAmp PCR protocol, the concentration of each
deoxynucleoside triphosphate (dNTP) is 200 µM. For best base incorporation,
keep the four dNTP concentrations balanced and above the estimated Km of each
dNTP (10–15 µM).
Note
Some applications may deviate from these standards for dNTP
concentrations.
Lower dNTP concentrations should not significantly affect yield of PCR
amplification product.
Magnesium Ion Concentration
For most PCR amplifications, you can relate yield and enzyme fidelity to the free
Mg++ concentration (free Mg++ concentration is the concentration of Mg++ minus
the total dNTP concentration added to the reaction solution). When using
AmpliTaq DNA Polymerase, too little free magnesium ion results in little or no
PCR product, and too much free magnesium ion can produce a variety of
unwanted by-products.
Magnesium ion concentration generally has the most significant effect on
amplification yield and specificity. To identify the optimal magnesium ion
concentration; that which produces the highest yield of a specific PCR product,
you can perform the following experiment. In the presence of 0.8 mM total dNTP
concentration, run a series of 0.5 mM magnesium chloride concentration
increments over a 1–4 mM range and identify the optimal concentration.
Primer Concentration
PCR primers are oligonucleotides, typically 15–30 bases long, hybridizing to
opposite strands and flanking the region of interest in the target DNA. When
choosing two PCR primers, never choose primers that contain bases
complementary to themselves or with each other. Especially avoid primers that
show complementarity at the 3' and 5' ends. These often promote formation of
product artifacts called primer-dimers or primer-oligomers.
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To avoid problems with the internal secondary structure of primers or long
stretches of any one base, choose primers with a 40–60% G+C content. Also, avoid
using primers that bind to a target region of secondary structure For instance,
RNA sequences often have regions of looped secondary structures. Primers will
have difficulty annealing to these target sequence.
By adding non-template complementary 5' extensions to primers you can
facilitate a variety of useful post-amplification manipulations of the PCR product
without adversely affecting the amplification process. These 5' extensions can be
restriction sites, or promoter sequences.
Template Concentration
PCR templates may be single or double-stranded DNA or RNA. If your starting
sample is RNA, then your template can be:
•
Total cellular RNA
•
Poly (A+) RNA
•
Viral RNA
•
tRNA
•
rRNA
To prepare first-strand cDNA prior to conventional PCR amplification, you can
include a reverse transcriptase such as MuLV or recombinant Thermus thermophilus
(rTth) DNA Polymerase. For RNA templates with high G+C content or complex
secondary structure, the high-temperature reverse transcriptase activity of
thermostable rTth DNA Polymerase is effective.
If your starting sample is DNA, use nanogram amounts of cloned template,
microgram amounts of genomic DNA, or up to 20,000 target copies to start
optimization trials.
Even very low template concentrations (mRNA from tens of cells, DNA from
single cells or individual genomes) are often sufficient for PCR amplification.
Enzyme Concentration
AmpliTaq DNA Polymerase is the recombinant form of Taq DNA Polymerase. It
is obtained by expressing the Taq DNA Polymerase gene in an E. Coli host. Like
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native Taq DNA Polymerase, it lacks endonuclease and
3'-5' exonuclease activities, but has a 5'-3' exonuclease activity. AmpliTaq DNA
Polymerase is the enzyme used for the majority of PCR applications.
For most PCR applications with a 100 µL reaction volume, 2.0–2.5 units of
AmpliTaq DNA Polymerase are recommended. The enzyme can be added
conveniently to a fresh Master Mix prepared for a number of reactions, thereby
avoiding the tedium and possible accuracy problems associated with adding
individual 0.5 µL enzyme aliquots to each tube.
Recombinant 94-kDa Thermus thermophilus (rTth) DNA Polymerase is able to
quickly and efficiently reverse transcribe RNA to cDNA in the presence of MgCl2,
after chelation of the magnesium ion with EGTA. RTth DNA Polymerase can thus
be used as both a thermostable reverse transcriptase and as a thermostable DNA
polymerase in successive reactions in the same tube.
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Performing Hot Start PCR
Competing side reactions can become a major obstacle to routine, sensitive and
specific PCR amplification. Examples of side reactions include amplification of
non-target sequences in background DNA (mis-priming) and primer
oligomerization. These reactions usually occur during pre-PCR setup when you
mix together all reactants at room temperature.
In the Hot Start™ technique, you add reagents to the reaction tubes so that all
reactants do not mix until they reach a temperature high enough to suppress
primer annealing to non-target sequences. You can perform either a manual Hot
Start or an AmpliWax™ PCR Gem-mediated Hot Start.
To perform a manual Hot Start:
Note
Although manual Hot Start can increase specificity and yield, it is
inconvenient and you may encounter reproducibility and contamination
problems.
1.
At room temperature, mix all reactants except AmpliTaq DNA polymerase
below a mineral oil cap.
2.
Load all tubes into a GeneAmp PCR instrument system.
3.
Define temperature control parameters so that the temperature rises to
70–80˚C.
4.
Add AmpliTaq DNA polymerase enzyme to each tube, changing pipet tips
after each sample.
To perform AmpliWax PCR Gem mediated Hot Start:
E-8
1.
At room temperature mix all reactants except AmpliTaq DNA polymerase
in each reaction tube.
2.
Add a single AmpliWax PCR Gem to each reaction tube.
3.
Define temperature control parameters so that the temperature rises to
70–80˚C for 5 to 10 minutes, then cools to room temperature. This creates
a wax barrier over the aqueous layer.
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4.
Add the omitted reagent(s) and the test sample above the solid wax layer.
5.
Create a PCR thermal profile as follows:
a.
Rapidly heat samples to the first denaturation temperature.
This melts the wax layer, denatures the DNA template, and creates
enough thermal convection to assure complete mixing of all PCR
components under the melted wax. The wax also serves as a vapor
barrier during cycling.
b.
6.
Program temperature control parameters for conventional thermal
cycling.
Run the PCR.
After thermal cycling ends, the wax forms a solid shield, preventing
spillage and evaporation.
7.
For post-PCR analysis, you can penetrate the wax layer with a pipet tip and
withdraw the PCR product.
8.
Reheat to seal for long-term storage.
Note
March 2001
AmpliTaq Gold DNA polymerase is a modified form of AmpliTaq DNA
polymerase specifically designed for Hot Start PCR. AmpliTaq Gold is
provided in an inactive state and regains activity through a pre-PCR
heat step. Contact Applied Biosystems for more information about
AmpliTaq Gold.
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Setting Temperature Control Parameters
Temperature control parameters are the temperature values and hold times you
specify for a PCR thermal profile. Table E-2 shows guidelines you can follow when
considering the effects of different PCR parameters on the outcome of a PCR
reaction.
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Table E-2. How Changing Temperature Control Parameters Affects the PCR
Temperature Control Parameter
PCR Performance
Raising the denaturation
temperatures (up to 96˚C)
Amplifies GC-rich templates
Lowering annealing temperatures
Increases yield, but may reduce
specificity
Varying the annealing or
denaturation temperatures in each
run. For example, six runs with
annealing temperatures that vary by
2˚C per run.
Increases chances of annealing
Setting each denaturation, annealing,
and extension step to at least 15
seconds (preferably 30 seconds).
Allows complete extension of PCR
product
Using the autoextension function of a
thermal cycler, to vary the parameters
to allow longer extension times in the
later cycles.
Increases yield
DNA denaturation is the critical step in the GeneAmp PCR process and is often
the focus of attention if PCR experiments fail. The practical range of effective
denaturation temperatures for most samples is 94–96˚C.
Always allow sufficient time for thermal equilibration of the sample at this hightemperature plateau.
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Avoiding Contamination
PCR protocols can be very sensitive to contaminants in the DNA, as well as to DNA
degradation that occurs before or during the purification process.
Note
Although many protocols have been published recently that describe
“simple” or “fast” extraction or purification methods, you should carefully
evaluate any changes or improvements in extraction or purification
methods. Also, be sure that the physical and chemical condition of the
sample itself is adequate for the intended labeling and assay method.
Sample contamination when preparing or analyzing samples can impair the PCR
process, and lead to poor results.
Preventing PCR Product Carryover
Carryover of PCR products from previous amplifications contaminates samples,
resulting in false–positive amplifications. Adopting the following laboratory
practices can help you minimize the likelihood of PCR product carryover:
E-12
•
If possible, use positive displacement pipets.
•
Always run a negative control to ensure that target DNA from previous
experiments does not contaminate your samples.
Note
A negative control contains no DNA. The negative control contains only
the DNA diluent (usually water or buffer).
•
For each run, use the same reaction composition and samples in the same
tubes, but after each run transfer the product to a unique product tube.
•
Physically separate reactions prior to and following amplification. For each
reaction, use separate sets of devices such as automatic pipetters,
disposable pipet tips, microfuge tubes, and gloves.
•
Use identical pipetting techniques for each run, but handle pre- and postPCR solutions with separate dedicated pipetters.
E Optimizing PCR
March 2001
Applied Biosystems
•
Aliquot reaction reagents so as to minimize the number of times you use a
particular stock solution.
•
Use AmpErase® UNG in reaction mixtures to prevent the subsequent reamplification of DU-containing PCR products.
Cleaning Up Spills on Work Surfaces
If you do spill samples on your work surface, use a cloth soaked with 50% bleach
to clean your bench top, thermal cycler sample blocks, or any other work surfaces
contaminated with nucleic acids.
IMPORTANT
March 2001
When cleaning the sample block of a thermal cycler, refer to the
instrument manual for proper cleaning instructions.
E Optimizing PCR
E-13
Applied Biosystems
Analyzing Post-PCR Products
If your PCR does not produce enough product to analyze, check the following:
•
Did you add the right amount of enzyme to your reaction mixture?
•
Did you use the right size GeneAmp or MicroAmp Reaction tubes for the
Applied Biosystems thermal cycler you are using?
•
Did you check the chemical integrity of the primers?
Suggestions for improving yield:
E-14
•
Pre–incubate samples at 95˚C for 5 to 10 minutes in the absence of enzyme.
This inactivates harmful proteases or nucleases in the sample. Preincubation also ensures complete denaturation of complex starting
templates.
•
Consider performing the Hot Start technique (See Performing Hot Start
PCR on page E-8).
E Optimizing PCR
March 2001
Applied Biosystems
Glossary
This glossary defines special terminology
used in the ABI PRISM 373 DNA Sequencer
with XL Upgrade User’s Manual. The terms
are listed in alphabetical order. Many terms
are defined in the text of the user’s manual,
so if you do not find a term here, check the
index.
electropherogram – a four-color
representation of a sequence, showing peaks
that represent the bases. The term is used
interchangeably with chromatogram in this
manual. An electropherogram is generated
by the Sequence or GeneScan Analysis
applications.
beam stop – laser beam safety stop. This is the exporting – storing the contents of selected
bar that prevents outside exposure to the
sequences in a file other than the associated
laser beam when it is scanning the gel.
data file.
channels – divisions across the read region of
a gel where the data collection software
samples the data. The number of wells in the
loading comb used determines the
approximate number of channels assigned
per lane of the gel (for instance, with a 36well comb, one lane is approximately five
channels). When the Sequencing Analysis
software tracks a gel, it places the tracker line
for each lane in the channel showing the
strongest fluorescent signal. The data from
that channel and the adjacent channel on
each side (total of three) are averaged to
determine the raw data for the sample file.
Firmware Image – a file containing the
executable software needed to run the
Translation Processor (TP). This code is
automatically sent to the TP by the
ABI PRISM 373XL collection software
running on the Macintosh, if the TP
indicates it has no firmware.
gel casting comb – a specially formed piece of
Mylar or Valox used to create wells or a single
slot at the top of the gel during
polymerization. For Sequencing
applications, use the sharks-tooth comb after
the single slot is formed by the gel casting
comb.
Classic – This configuration includes 370s
with Macintosh upgrade and the 373A.
Classic models have electrophoresis
chambers which support 24-cm WTR
distances.
gel image – the colored display of the Gel file
that appears on the computer screen. To
decrease the size of the file, you can use the
GelDocII program to remove the image from
the file. If you do so, you cannot display the
data point – The data collection software
gel image on the screen. If you remove the
samples data 194 or 388 times as it scans
image from the file, you can also use the
across the gel. Each “sampling” is stored as a GelDocII program to rebuild the image.
data point. A data point is also represented by
grid - a spreadsheet-like display used for
one line on the Map or Gel display.
entering data in tabular format. The Sample
March 2001
Glossary
G-1
Applied Biosystems
Sheet and several data collection windows
display grids for entering Sample
information.
INIT – a Macintosh system extension that
expands the capabilities of the system
software. Examples are printer drivers and
programs that add features to the Finder or
hygroscopic - readily absorbing moisture
the system software. INITs are stored in the
extensions folder inside the System folder.
image – see firmware image, gel image.
Do not use CDEVs and INITs other than
Instrument file – a file used to adjust for the those shipped with your system.
spectral overlap between the fluorescent dyes
used on the ABI PRISM 373 DNA Sequencer IUB code – alphabetic character
with XL Upgrade. A mathematical matrix of representing the occurrence of mixed bases
at a given position in a sequence. Originally
the spectral overlaps is created and the
defined by the International Union of
inverse matrix is used to correct the data
during analysis. The file is stored in the ABI Biochemistry. The following table contains a
list of IUB codes, the mixed bases they
folder in the System folder.
represent, and a listing of the complements.
IUB Codes and their complements
IUB Codes
R = A or G (puRine)
A
T
S
W
C - Cytidine
Y = C or T (pYrimidine)
C
G
W
S
G = Guanosine
K = G or T (Keto)
G
C
T = Thymidine
M = A or C (aMino)
T
A
B
V
B = C,G, or T
S = G or C (Strong— 3 H bonds)
D
H
D = A, G, or T
W = A or T (Weak—2 H bonds)
R
Y
H
D
H = A, C, or T
N = aNy base
Y
R
V
B
K
M
N
N
V = A, C, or G
IUPAC – International Union of Pure and
Applied Chemistry. IUB codes are also
referred to with this acronym, since IUPAC
adopted the codes as a standard.
length – the length of a sequence is the
number of characters it contains.
LED – light emitting diode. In this manual,
this term refers to the status-indicator lights
G-2
Complements
A = Adenosine
Glossary
on the front and the back of the instrument.
In general, this refers to a “semiconductor
diode that converts applied voltage to light
and that is used in digital displays”, such as
calculator and some computer displays (The
American Heritage Dictionary, Second College
Edition, Houghton Mifflin Company:
Boston/New York,1991; p 721).
March 2001
Applied Biosystems
Leon – this Model 373 configuration has an
electrophoresis chamber which supports 6-,
12-, and 24-cm WTR distances.
separation distance – the length from the
wells of the gel to the read region of the gel.
Also called the well-to-read or WTR.
matrix file/multicomponent matrix – a file
used to adjust for the spectral overlap
between the fluorescent dyes used on the ABI
PRISM 373 DNA Sequencer with XL Upgrade.
A mathematical matrix of the spectral
overlaps is created and the inverse matrix is
used to correct the data during analysis. The
file is stored in the ABI folder the System
folder.
sequence – a linear series of characters. The
characters are displayed in rows from left to
right. More specifically, a sequence is a series
of nucleotide base characters that represent a
linear DNA sequence, or a series of amino
acid characters that represent a protein
sequence.
module – a file that provides instructions
about conditions of operation to the
instrument. You might use three different
modules during a typical run to specify
conditions for plate check, pre-run, and the
run itself.
PMT – photomultiplier tube.
sequencing reactions – the chemistry
performed to incorporate fluorescent dye
labels into DNA extension products. The
chemistries and kits supplied by Applied
Biosystems for performing such reactions are
described in the ABI PRISM 300Sequencing
Chemistry Guide.
settings – choices that you specify in the data
collection software about the parameters
used for data collection.
read region – The region of the glass plates
sharks-tooth comb – Mylar or Valox device
where the laser scans. The location of this
area depends on the instrument. See Figure used to form wells for a sequencing gel. It is
inserted after the gel has polymerized with
6-3. and Figure 6-3. on page 2-6.
the gel casting comb in place. The casting
sample files – data files produced by the
comb is removed, leaving a slot into which
Analysis Software associated with the ABI
the sharks-tooth comb is inserted.
PRISM 373XL DNA Sequencer. These files
contain data produced by the instrument: a square-tooth comb – specially formed piece
sequence of base calls, peak locations, and an of Mylar or Valox used to create squareelectropherogram. The original data in
shaped wells at the top of a gel during casting.
sample files is always maintained in its
pristine state (as it came from the ABI PRISM Stretch - this Model 373 configuration
supports 6-, 12-, 24-, 34-, and 48-cm WTR
373 DNA Sequencer with XL Upgrade).
When you save changes you have made to the distances and is easily identified by its
extended door.
sequence, they are stored to a copy of the
original data called the editable data.
tared – counterbalanced.
March 2001
Glossary
G-3
Applied Biosystems
text files – type of simple file produced by all
Macintosh word processing programs, and
many other programs.
Translation Interface Processor– this device,
which sits on top of the computer’s CPU,
serves as the communication link and data
buffer/translator between the ABI PRISM 373
DNA Sequencer with XL Upgrade and the
Power Macintosh.
well-to-read length (WTR) – the distance
from the wells of the gel to the laser scanning
(read) region of the gel; separation distance
for a run. The length of the glass plates is
usually expressed as the well-to-read length
(for instance, when you prepare a gel using
34-cm plates, the separation distance
between the sample loading wells and the
read region of the gel is 34 cm).
G-4
Glossary
March 2001
Applied Biosystems
Index
difference from ABI 373 1-20
general description 1-9–1-13
0.2 mm gel spacers, using when assembling
power switch 6-17
the plates 2-15
status lights 6-17
0.3 mm gels casting comb
system hardware 6-3–6-5
48 cm combs 2-26
using for sequencing and
10 mM Tris-Cl (pH 8.0), 1 m M EDTA (pH
GeneScan 1-10–1-11
8.0) B-11
See Also computer; maintenance
24 cm BaseSprinter
ABI PRISM 373 with XL upgrade
preparing 4.75% gel 2-19
controlling manually 5-59–5-60
34 cm Full Scan gel
data flow diagram 5-72
preparing 4.75% gel 2-19
detection described 2-9
373 DNA Sequencers
firmware 6-18
configurations 1-4
general default settings 3-20, 4-23
difference from ABI PRISM 373 with XL
general description
upgrade 1-20
how it works 1-12–1-13
373 with XL upgrade 23
instrument maintenance 8-3–8-7
4% acrylamide-urea gel solutions 2-19, 2-25
instrument status and control 6-19–6-20
4.75% acrylamide-urea gel solutions 2-19
Macintosh vocabulary and operations 7-6
48 cm gel
programs that come bundled with 1-16
casting 2-23–2-24, 2-26–2-27
setting up 3-6–3-38, 4-9
preparing 4.0% acrylamide gels 2-25
software provided 7-5
48 cm run
system hardware 6-3–6-5
applying the Bind-Silane
throughput 1-14
compound 2-14, 2-22
translation processor status and
preparing glass plates 2-22
control 6-17–6-19
6% acrylamide-urea gel solutions 2-19
using for sequencing and GeneScan 1-11
About ABI PRISM 373 with XL
upgradecommand 5-5
abbreviations, table of B-10–B-11
acrylamide
ABI folder
denaturing gels described 2-9–2-12
files located in 5-73
effect of impurities on
See Sequencing Analysis program
sequencing 2-3–2-4
ABI PRISM 373 DNA Sequencer with XL
gels
upgrade
electrophoresis 1-12
system hardware 6-3–6-9
recipes B-5
ABI PRISM 373 with XL upgrade 15
troubleshooting 8-3
clearing random access memory 7-8
Symbols
A
March 2001
Index
I-1
Applied Biosystems
polymerization 2-3
preparing stock solution 2-11
safety warning 2-4, 2-9, 2-18
solution for gel 2-18–2-19
stock solution purchase 2-4, 2-10
storage 2-4
acrylamide-urea gel solutions
recipes for 2-18–2-19, B-5
See Also acrylamide
acrylic acid, hydrolysis product 2-4
adding
software programs 7-10
After Dark®
compatibility with the instrument
system 7-10
air bubbles
effect on gel quality 2-7
under gel plates in buffer chamber 4-41
See Also quality, factors affecting gel quality
Alconox™
using to wash plates 2-13, 3-48, 4-51
alerts, red 5-57
alignment algorithms for DNA 1-18
alignment brace, marking on
Classic configuration 4-36
Leon configuration 3-36, 4-38
Stretch configuration 4-40
amber LED 6-19
ammonium persulfate. See APS
AmpErase UNG E-13
See Also carryover of PCR products
amplification, unsuccessful 4-58, 4-59
AmpliTaq® DNA Polymerase 4-58, E-6
commonly used 1-11
reducing concentration to improve data
quality 4-57
AmpliWax PCR Gem E-8
I-2
Index
analysis
after analysis, automatic
printing 3-25, 4-53, 5-67
analyzing data electropherograms 3-51
automatic
analysis 3-50–3-51, 4-53, 5-24, 5-27
and sample sheet 5-32
customize settings before run 4-8, 4-19
default Base Caller for automatic
analysis 3-16
further analysis of processed
data 1-17–1-18
recording sample
information 3-21–3-22, 5-32
Sequencing Analysis program 5-75
setting up automatic analysis 3-15
use separate sample sheets for sequence
or fragment analysis 3-21
using a setting other than default 3-5
See Also GeneScan
analysis software, setting up
GeneScan mode 4-19
recommended amount of RAM 7-3
Sequencing mode 3-16
analyzing 3-50–3-51
electropherograms 3-51
in GeneScan mode 4-53
post-PCR products E-14
wavelengths of the dyes being
detected 3-50
annealing temperature E-11
increasing to improve data quality 4-57
Apple menu 5-5
APS 2-3
adding to cast the gel 2-20
changing concentration, effect of 2-7
preparing gels 2-12
storage 2-5
archiving 4-65–4-66
March 2001
Applied Biosystems
backing up data to magnetic
tape 3-53, 4-66
before optimizing your disk 7-12, 8-7
files 5-66
created during a run
GeneScan mode 4-65–4-66
Sequencing mode 3-52–3-53
other files 3-53, 4-66
See Also saving
Auto Analyze checkbox 3-50–3-51
automatic analysis after analysis 4-53
completing the Sequence Run window for
a pre-run or run 5-43
Auto Print checkbox 3-25, 4-53
automatic printing after
analysis 4-29, 5-45, 5-67
completing the Sequence Scan window
for a pre-run or run 5-43
AutoAssembler software™ 1-17, 1-18
autoextension, uses of E-11
automatic analysis. See analysis
automatic lane tracker 3-41
autoradiogram
reconstructed data displayed in Gel
window 1-13
See Also Gel window
B
backing up. See archiving
bands, smeared or fuzzy 4-57
Base Callers
default for automatic analysis 3-16
base spacing 1
baseline subtraction, processing data 1-16
bases
colors in real-time displays 5-53, B-9, C-4
positions on electropherogram 3-51
BaseSprinter mode 6-12–6-13
battery backup 6-17
March 2001
Index
beam-stop bar 1
installing 4-13
Bind-Silane
applying the compound to the
plate 2-14, 2-23
bis-acrylamide 2-4
effect of impurities on sequencing 2-3
polymerization 2-3
safety warning 2-4, 2-9, 2-18
storage 2-4
buffer
affect of impurities 2-6
flush wells before loading 4-41
loading for better accuracy 3-40
recipes 2-10–2-11
buffer chambers 6-10
removing air bubbles 4-41
See Also individual sequencer
configuration
buttons
Cancel button 5-46
color, using to change color 5-30
Execute button (manual control) 5-59
Pause button 5-46
pausing to load additional
samples 3-43
PreRun button 3-39, 4-41, 5-45
to control instrument 3-23, 4-27, 5-41
C
calibration file
checking and adjusting 5-61–5-64
Cancel button 5-46
Cancel Run 5-9
using the Cancel button to stop a pre-run
or run 5-46
using to stop a run before it is
finished 5-48
carryover of PCR products E-12–E-13
I-3
Applied Biosystems
evidence of 4-56
casting comb. See gel casting comb
casting gels 2-20
48 cm 2-23–2-24
48 cm gel 2-26–2-27
inserting the gel casting comb 2-20
See Also gels
channels 1
numbers in lanes 3-29–3-31
chemical hazard
formamide 3-40, 4-42
reagents and supplies for gels 2-11
See safety warnings
chemistry
associated with dye set 5-52
determines color on real-time
display 5-52
sequencing C-3
chromatograms. See electropherograms
Classic, Model 373 DNA Sequencer
configuration 1-4, 1
completing instrument
setup 3-35–3-36, 4-36–4-37
model specific notation 3-8, 4-11
preparing the electrophoresis
chamber 3-9, 4-12–4-13
replacing lower electrode 8-6–8-7
replacing upper buffer chamber 8-4–8-5
cleaning
after a run
in GeneScan mode 4-50
plates, comb, and spacers 4-51
in Sequencing mode 3-47
instrument accessories 8-3
plates
in GeneScan mode 4-31–4-32
in Sequencing mode 3-28–3-29
sample block of thermal cycler E-13
up spills in work area E-13
I-4
Index
Clear command 5-8
client-server, upgrade for the AutoAssembler
Sequence Assembly Software 1-18
Close command 5-6
closing windows 5-10, 5-49, 5-68
codes, dye indicator defaults 5-30
colors
bases in real-time displays 5-53
in electrophoresis history window 5-57
in real-time displays C-4, D-4
setting defaults for data display and
printing 5-29
using Color button to change color 5-30
wavelengths of the dyes being
detected 3-50
combs 6-7–6-9
compatibility with spacers and casting
comb 2-15, 3-34
table of combs and run modes B-9
See Also gel casting comb
comprehensive record of errors,
status 3-45, 4-48
computer
ABI PRISM 373 with XL upgrade
system 1-9
adding software programs 7-10
installing the software 7-4–7-5
log of conditions 5-74
Macintosh vocabulary and operations 7-6
maintenance 7-7–7-12
rebuilding the desktop 7-5
software provided 7-5
special software for data collection and
analysis 7-5–7-6
system requirements 7-3
See Also Macintosh
computer system. See computer
concentration
of initiator and polymerization 2-6, 2-7
March 2001
Applied Biosystems
contamination E-12–E-13
cleaning plates 3-28–3-29, 4-31–4-32
cleaning up spills E-13
preventing PCR product
carryover E-12–E-13
using distilled, deionized water to avoid
contamination of reagents 2-6
See Also marking the alignment brace;
samples
control reactions
evaluating quality of data 4-54
inconsistent results with 4-60
COP assay
using GeneScan to perform quantitative
gene analysis 1-17
Copy command 5-8
corrupted memory 6-18
CP Optimizer
using to maintain your hard disk 7-11
creating
GeneScan analysis sample
sheet 5-36–5-37
GeneScan matrix file D-6–D-15
creating the matrix file D-10–D-12
matrix standards D-6
preparing, loading and electrophoresing matrix
standards D-6–D-9
GeneScan sample sheet 4-24–4-26
icon for creating Run folder 5-40
Instrument file C-5
Run file for GeneScan mode 4-26–4-29
sample sheet 3-21–3-22, 5-36–5-37
Sequence sample sheet 5-34–5-35
crossover between color channel
(troubleshooting) 4-61
current (voltage), problems with 4-64
Cut command 5-8
March 2001
Index
D
data
analysis software 1-16–1-18
appearing as on autoradiogram 1-13
archiving 3-52–3-53
characteristics of good data 4-55
characteristics of poor data 4-56
color in real-time displays D-4
evaluating quality of 4-58–4-64
general description of data analysis 1-13
interpreting 3-50–3-51
printing 1-13
real time observation of data 1-12, 3-45
saving 3-52, 4-65–4-66
software for analysis 1-16–1-18
See Also viewing
data collection
data collection run log error
message 8-16
getting program information 5-5
menu commands 5-5–5-11
PMT gain and calibration file 5-61–5-64
preparing for a run 5-12
program files 5-70–5-76
files located in the system
folder 5-76
input and output files 5-73–5-76
output files 5-74–5-75
temporary files located in the system
folder 5-76
quitting the program 5-68
setting preferences for data collection
program 5-14–5-29
starting Data Collection software
in GeneScan mode 4-20–4-29
in Sequencing mode 3-17
temporary files created in the system
folder 5-76
I-5
Applied Biosystems
using the data collection
program 5-12–5-13
data point 1
database, exporting files for 5-67
DataUtility software
bundled with Sequencing Analysis
program 1-16
using to make and copy matrices 7-5
ddNTP, defined B-10
defaults 3-19–3-20, 4-22–4-23
Base Caller for automatic analysis 3-16
file names in Sequencing
mode 3-19, 4-22
general settings 5-27–5-29
saving module settings as
defaults 3-18–3-19
setting
for GeneScan Analysis
applications 5-25–5-31
for Sequencing Analysis
applications 5-21–5-24
table of ABI PRISM 373 with XL upgrade
module default values B-6
definitions of terms 1–4
defragmenting your hard drive 7-7–7-10
deionizing resin
preparing acrylamide stock solution 2-11
preventing breakdown 2-9
denaturing gels
changing temperatures affects the
PCR E-11
preparing 2-9–2-12
See Also gel
deoxynucleoside triphosphate (dNTP)
optimizing PCR reaction E-5
detection
ABI PRISM 373 with XL upgrade
description 1-12
affected by gel fluorescence 2-9
I-6
Index
dH2O, defined B-10
diffuse bands, resulting from Acrylic acid 2-4
digitized output signal 1-12
Disk Express II
using to maintain your hard disk 7-11
using to optimize the hard disk 7-9
DiskTools, using to defragment your hard
drive 7-7–7-10
displays, in real-time 5-52–5-58
filters and color guide 5-52–5-54
using
the Electrophoresis History
window 5-57–5-58
the Gel window 5-56
the Scan window 5-55
Dithiothreitol B-10
DNA
alignment using Sequence
Navigator 1-18
assembling using AutoAssembler
software 1-17
electrophoresis and detection 1-12
incorporating dye labels 1-11
mitochondrial, using Sequence Navigator
to screen sequences 1-18
optimal quantities for sequencing 3-5
PCR template concentration E-6
resuspending 3-40
sizing and quantitating
fragments 1-9, 7-5
table listing sequencing resuspension and
loading volumes 3-40
template concentration E-6
using a negative control E-12
DNA Sequencing Analysis software 1-16
See Also Sequencing Analysis program
DNA Thermal Cycler
tube types E-4
DNA Thermal Cycler 480
March 2001
Applied Biosystems
tube types E-4
document icons
in run window 3-23, 4-27, 5-40, 5-41
DTT (Dithiothreitol) B-10
dyes
analyzing wavelengths of the dyes being
detected 3-50
dye indicator defaults 5-29
fluorescent dyes for labeling 1-12
indicator defaults 3-20, 4-23
table
dye set and filter sets by
chemistry B-9
dyes and filter set color displays D-5
DyeSet/Primer
choosing in sample sheet 5-35
file naming conventions 5-34–5-35
setting default for sample sheet 5-21
E
Edit menu 5-8
editing, sample sheet 5-37–5-39
EDTA
defined B-10
resuspending DNA 3-40
electrical shock. See safety warnings
electrodes, replacing 8-3
electropherograms 1
reading 3-51
setting default colors for 5-29
electrophoresis
ABI PRISM 373 with XL upgrade
description of how instrument
works 1-12
chamber, setting up for sequencing 3-12
current and voltage during a run 5-50
power supply
about 6-10
March 2001
Index
displaying the set and actual
values 5-57
pre-running to check the system 3-39
replacing cables and electrodes 8-3
troubleshooting table for GeneScan
mode 4-58
Electrophoresis History window
command 5-11
setting scale 5-58
using to view instrument status in
real-time 5-57–5-58
enzyme concentration E-6–E-7
errors
data collection run log errors 8-16
log file 3-45, 4-48
messages 7-11
using the Log file 5-51–5-52
Excel®, exporting the sample sheet to 5-74
Execute button 5-59
exporting 5-7, 1
sample sheet information 5-33
using to save the contents of a window
grid 5-67
extensions
turning off when restarting 7-10
extraction procedures 4-58
F
Factura software 1-17
felt-tipped pens, using. See marking the
alignment brace
File menu 5-6–5-7
files
.seq files 5-75
ABI folder, files located in 5-73
archiving 5-66
automatically change generated file
names 5-20
Data Collection program files 5-70–5-76
I-7
Applied Biosystems
ensuring disk space for automatically
created
files 3-27, 3-42, 4-30, 4-45, 4-46
importing and exporting 5-67
input files in the Data Collection
program 5-71–5-74
located in the system folder 5-76
locations on computer 5-71
output files in the Data Collection
program 5-74
printing 5-66–5-67
removing non-essential files from hard
disk 7-9
resetting global serial number (for file
names) 5-27
Sample file format compatible with other
programs 1-16
saving 5-65
temporary files 5-76
See Also run file; sample sheet
Fill Down command 5-8
filter sets
color displays, table of D-5
filter set table for GeneScan 5-53–5-54
filter wheel and data
display 6-13–6-15, C-3–C-4
GeneScan matrices D-4–D-5
firmware 6-18
clearing if memory corrupt 6-18
image, defined 1
fluorescence
checking the plates for 3-27–3-38
dye-labeled primers 1-11
spectra
matrices C-3
overlap corrected with matrices D-3
See Also dyes
flushing wells before loading 4-41
I-8
Index
folders
changing data storage folders in
preference settings 5-17–5-18
creating Run folder 5-40
default location 3-19, 4-22
diagram, locations on computer 5-71
forensics applications, evaluating the quality
of the data 4-54
formamide
chemical hazard 3-40, 4-42
resuspending DNA 3-40
using in empty wells 3-42, 4-44, 4-45
format
log file 5-51
tab-delimited text 5-67
fragment analysis
sample sheet defaults 5-25
sizing and quantitating 1-9, 7-5
use separate sample sheet 3-21, 5-32
See Also GeneScan
free radical, factor that affect gel quality 2-7
Full Scan mode 6-12
G
gel
casting 2-20–2-21
cleaning plates after run 3-48
color guide for gel image displays B-9
completing
GeneScan Run window for a pre-run
or run 5-43–5-45
Sequence window for pre-run or
run 5-41–5-43
detection described 2-9
determining which lanes in a gel are
contaminated 3-29–3-31
factors that affect gel quality 2-6
matrix formation 2-3
polymerization mechanism 2-3
March 2001
Applied Biosystems
preparing
the acrylamide-urea
solution 2-6, 2-18–2-19
the gel for loading in GeneScan
mode 4-9–4-10
the glass plate 2-13–2-14
to mix sequencing gel 2-11–2-12
pre-running the gel 3-39
reagents
for stock solutions 2-10–2-11
purity 2-18
reagents and supplies 2-11–2-12
reducing the size of the gel image file 1
removing the gel from the instrument
in GeneScan mode 4-50–4-51
in Sequencing mode 3-47–3-48
run readings under normal
conditions 5-51
scanning the plates for
fluorescence 3-27–3-38, 4-30–4-31
temperature status during run 1-12, 5-57
tips for preparing gels for long runs 2-20
troubleshooting 8-14–8-15
using for consistent results 2-8
gel cassette
mounting glass plates 2-15–2-17
gel casting comb 1
48 cm gels 2-26
air bubbles trapped between comb and
solution 2-7
compatibility with combs and
spacers 2-15, 3-34
inserting 2-20
See Also square-tooth comb; Sharks-tooth
comb; gel, preparing to mix
sequencing gel
gel files 5-74
acrylamide gel recipes B-5
and GelDoc II software 1-16
March 2001
Index
archiving 3-52, 4-65
removing file from hard disk 7-9
saving 3-52, 4-65, 5-66
using GelDocII 7-5
gel image 1
command 5-11
gel plate rest
adjusting 4-16
components of 3-13, 4-16
gel spacers 2-15, 6-7
compatibility with combs and casting
comb 2-15
if longer than the full length of the glass
plates 2-15
Gel window
viewing data 1-13
in real-time 3-45, 4-49
viewing instrument status 5-56
GelDocII 1-16
using on Gel files 7-5
using to reduce the size of the gel image
file 1
GeneAmp PCR System 2400
tube types E-4
GeneAmp PCR System 9600
misaligned lid 4-60
tube types E-4
GeneAmp Thin-Walled Reaction Tubes
See reaction tubes
GeneAssist, using to search biological
databases 1-18
GeneScan 1-17
analysis application, setting
defaults 5-25–5-31
creating a sample sheet 5-36–5-37
filter set table 5-53–5-54
flowchart of typical run process B-4
Genotyper software to convert data 1-18
matrices D-3–D-15
I-9
Applied Biosystems
creating a matrix file D-6–D-15
creating the file D-10–D-12
matrix standards D-6
preparing, loading and electrophoresing matrix
standards D-6–D-9
filter wheel and data
display D-4–D-5
Matrix Standard kits D-3
verifying quality of
matrix D-13–D-15
preparing samples 4-41–4-43
sample files created by 5-75
sample sheet
different from sequencing 3-21
run defaults 5-25
set analysis parameters before
run 4-8, 4-19
table of run times and filter sets B-8
template shipped with
system 3-17–3-19, 4-20–4-21
using on the ABI PRISM 373 with XL
upgrade 1-11
using on the ABI PRISM 373 with XL
upgrade 1-11
See Also GeneScan mode
GeneScan Analysis Software User’s
Manual 1-10
GeneScan mode
before you set up the instrument 4-5–4-8
experimental design
considerations 4-5–4-7
preparing samples 4-5
setting up the instrument 4-8
checking the plates 4-30–4-40
checking PMT setting 4-32–4-34
completing instrument
setup 4-36–4-40
I-10
Index
inserting the Sharks-tooth
comb 4-34–4-35
cleaning, after a run 4-50
evaluating the quality of the data 4-54
characteristics of good data 4-55
characteristics of poor
data 4-56–4-57
loading samples 4-43–4-47
preparing the sample
for electrophoresis 4-42–4-43
pre-running the gel 4-41
setting up the instrument 4-9–4-18
preparing the electrophoresis
chamber 4-11–4-18
for Classic
configuration 4-12–4-13
for Leon
configuration 4-13–4-14
for Stretch
configuration 4-11–4-18
preparing the gel for
loading 4-9–4-10
setting up the software 4-19–4-29
creating a Run file 4-26–4-29
creating the GeneScan sample
sheet 4-24–4-26
setting default parameters 4-22–4-24
setting up the analysis software 4-19
starting the Data Collection
software 4-20–4-29
troubleshooting table 4-58
GeneScan Run window
completing for pre-run or run 5-43–5-45
Genotyper™ software 1-18
getting started
quick start information 1-8
glass plates
about 6-6–6-7
alignment example 2-16
March 2001
Applied Biosystems
casting gel example 2-13
cleaning 2-13–2-14
after a run 3-48
previously used plates 2-14
drying plates 2-13
marking the outside to use the same
side 2-14, 6-7
mounting in cassette 2-15–2-17
non-fluorescence importance of 2-9
position in buffer chamber 3-13, 4-16
preparing
for gels 2-22
for GeneScan mode 4-9–4-10
for loading the gel 3-6
scanning the plates for
fluorescence 3-27–3-38, 4-30–4-31
temperature for gel formation 2-6
global
parameters 5-14
serial number, resetting 5-27
glossary 1–4
base spacing 1
beam stop 1
channels 1
Classic (Model 373 DNA Sequencer
configuration) 1
data point 1
electropherogram 1
exporting 1
gel casting comb 1
gel image 1
grid 1
hygroscopic 2
INIT 2
Instrument file 2
IUB (International Union of
Biochemistry) codes 2
IUPAC (international Union of Pure and
Applied Chemistry) 2
March 2001
Index
LED 2
length 2
Leon (Model 373 DNA Sequencer
configuration) 3
matrix files 3
modules 3
multicomponent matrix defined 3
PMT 3
read region 3
sample files 3
separation distance 3
sequence 3
sequencing reactions 3
settings 3
Sharks-tooth comb 3
square-tooth comb 3
Stretch (Model 373 DNA Sequencer
configuration) 3
tared 3
text files 4
Translation Interface Processor 4
WTR (well-to-read length 4
green graph 5-57
green LED 6-19
grid 1
H
hard disk
backing up files 3-52, 4-65
ensuring space for automatically created
files 3-27, 3-42, 4-30, 4-45, 4-46
maintenance 7-7–7-10
adding software 7-10
optimizing 7-9
programs 7-11
removing non-essential files 7-9
hardware 6-3–6-9
components 6-4
loading system 6-6–6-9
I-11
Applied Biosystems
scanning and detection system 6-11–6-16
See Also importing
the read region 6-12–6-13
installing
system overview 6-4–6-5
software 7-4–7-5
See Also troubleshooting
rebuilding the desktop 7-5
hazard. See safety warnings
the beam-stop bar 4-13
heat transfer plate, installing
instrument
GeneScan mode
changing connection port on
24-cm and 34-cm runs 4-16
Macintosh 5-27
24-cm runs only 4-37
control buttons 3-23, 4-27, 5-41
Sequencing mode 3-13, 3-36
controlling manually 5-59–5-60
help 1-22–1-28
copying software image to firmware 6-17
See also troubleshooting
hardware 6-3–6-9
heterozygote
components 6-4
screening using the Sequence Navigator
loading system 6-6–6-9
Sequence Comparison software 1-18
scanning and detection
HIV, screening of sequences 1-18
system 6-11–6-16
homozygote 4-61
separation system 6-10
Hot Start technique
system overview 6-4–6-5
AmpliWax PCR gem mediated E-8
log of conditions 5-74
hygroscopic 2
maintenance
backing up files 8-7
cleaning instrument accessories 8-3
replacing, electrophoresis cables
icons
and electrodes 8-3
Run window icons 5-40
troubleshooting 8-8–8-16
small document on Run
clearing the memory 8-9–8-12
window 3-23, 4-27, 5-40, 5-41
gel troubleshooting 8-14–8-15
identifying samples 5-32
hardware/firmware 8-9–8-12
See Also samples
problems during a run 8-13
image, copying software image to
total reset of the instrument 8-8
firmware 6-17
troubleshooting software 8-16
importing 5-7, 5-68
setting up 3-6–3-38, 4-9
files 5-68
status and control 6-19–6-20
sample sheet information 5-33
electrophoresis history 5-57
See Also input and output files
status indicators 6-19–6-20
Inherit Sequence Analysis System 1-17–1-18
status lights 6-17
INIT 2
location and use on the
initiator concentration 2-7
instrument 6-19
and polymerization 2-6, 2-7
instrument and matrix files 6-15–6-16
input and output files 5-73–5-76
I
I-12
Index
March 2001
Applied Biosystems
Instrument file 2
creating C-5
lost file and no backup C-9
making the first matrix C-8–C-9
sequencing matrix file C-3
See Also matrix files
Instrument menu 5-9
starting a pre-run or run 5-48
instrument status, viewing 1-12
data in real-time 5-52–5-58
filters and color guide 5-52–5-54
using the Electrophoresis History
window 5-57–5-58
using the Gel window 5-56
using the Scan window 5-55
Run file for the current run 5-58
using the Status window 5-50–5-51
International Union of Biochemistry. See IUB
codes
International Union of Pure and Applied
Chemistry (IUPAC) 2
interpreting sequencing data 3-50–3-51
ion-exchange resin. See deionizing resin
Ionic contaminants, acrylamide 2-4
IPTG
(Isopropyl-ß-D-thio-galactopyranoside)
B-10
IUB (International Union of Biochemistry)
codes 2
IUPAC (International Union of Pure and
Applied Chemistry) 2
K
kits
Matrix Standard kits D-3
primer extension 1-11
March 2001
Index
L
lane
channel numbers in 3-29–3-31
moving information to a different
lane 5-39
specifying sample
in 3-21, 4-24, 4-26, 5-32
tracking
affected by air bubbles 2-7
verifying D-10
using the automatic lane tracker 3-41
laser
general function during the
electrophoretic process 2-9
hazard warning 3-28, 4-31
laser/scanner assembly, diagram of 6-11
region scanned, defined 3
using scan window to sweeps across the
gel 5-55
LEDs 2
status lights on the instrument 6-19
table of status 6-20
length, of a sequence 2
Leon, Model 373 DNA Sequencer
configuration 1-4, 3
completing instrument
setup 3-36–3-37, 4-37–4-39
model-specific notation 3-8, 4-11
preparing the electrophoresis
chamber 3-10–3-11, 4-11–4-14
replacing lower electrode 8-6–8-7
replacing upper buffer chamber 8-4–8-5
level gauge, preparing to mix sequencing
gel 2-12
light emitting diode. See LEDs
linear polyacrylamide, caused by catalytic
contaminants 2-4
linkage analysis, for use with Genotyper
data 1-18
I-13
Applied Biosystems
maintenance
loading
computer system
alternate wells with Shark’s-tooth
backing up files 7-12
comb 3-41
error messages 7-11
description of loading system 6-6–6-9
hard disk maintenance 7-7–7-10
flushing wells 4-41
programs for maintenance 7-11
samples
removing non-essential file 7-9
GeneScan mode 4-43–4-47
using hard disk 7-9
Sequencing mode 3-41–3-44
instrument
skipping multiple lanes 4-44
backing up files 8-7
using formamide in empty
cleaning instrument accessories 8-3
wells 3-42, 4-44, 4-45
replacing
using the lane tracker properly 3-42, 4-45
electrophoresis cables and
Log file
electrodes 8-3
command to view file 5-11
troubleshooting 8-8–8-16
log of computer conditions 5-74
clearing the memory 8-9–8-12
storing 3-52, 4-65
gel troubleshooting 8-14–8-15
using to view instrument status 5-51–5-52
hardware/firmware 8-9–8-12
long runs, tips for gel preparation 2-20
problems during a run 8-13
lower electrode replace. See individual
total reset of the instrument 8-8
instrument configuration
troubleshooting software 8-16
Make New Matrix dialog box example D-11
mAmp, in modules B-6
Macintosh
manual analysis with correct primer/dye
basic vocabulary and operations 7-6
set 5-35
clearing random access memory by
manual control 5-59–5-60
restarting before each run 7-8
displaying window for manual control of
Data Collection software described 6-18
functions during run 5-11
file locations 5-71
pausing or cancelling 5-60
instrument connection change 5-27
Manual Control window
See Also computer
using to control the instrument
magnesium ion
manually 5-59
concentration effects on PCR
manuals. See user’s manual
performance 4-62, E-5
reactions with rTth DNA Polymerase E-7 marking the alignment brace
on Classic configuration 4-36
magnetic tape drive, backing up
on Leon configuration 4-38
data 3-53, 4-66, 5-66
on Stretch configuration 3-37, 4-40
main menu 5-5
Master Mix, reason to create 4-57
M
I-14
Index
March 2001
Applied Biosystems
matrix
attaching new to Sample files D-13
DataUtility software 1-16
fluorescence spectra C-3
overlap corrected D-3
formation in gel 2-3
printing matrix values D-12
See Also matrix files, matrix standards
standards C-5
storing in ABI folder D-3
using standards C-5
verifying quality of matrix D-13–D-15
matrix files 3
choosing in sample sheet 5-35
creating C-5, C-9
making the first matrix for a new Instrument file C-8–C-9
matrix standards C-5
preparing, loading and electrophoresing matrix
standards C-6–C-8
GeneScan D-3–D-15
creating a matrix file D-6–D-15
creating the matrix
file D-10–D-12
matrix standards D-6
preparing, loading and electrophoresing matrix
standards D-6–D-9
filter wheel and data display D-4–D-5
Matrix Standard kits D-3
reasons to create a new matrix D-3
sequencing matrix files C-3
using DataUtility software to make and
copy matrices 7-5
See Also matrix; matrix standards
Matrix Standard kits provided by Applied
Biosystems D-3
Matrix Values window D-12
March 2001
Index
memory, clearing corrupted memory
symptoms of corrupted memory 8-9
menus
Apple menu 5-5
Edit 5-8
File 5-6–5-7
Instrument 5-9
main menu bar 5-5
Window 5-10–5-11
messages, errors 3-45, 4-48
metals, contaminating buffers 2-6
Mg++
See magnesium ion
MicroAmp Reaction Tubes
See reaction tubes
microgram B-10
Microsoft® Excel®
exporting the sample sheet to 5-74
migration, affected by
acrylamide contaminants 2-4
age of gel 2-8
mitochondrial DNA 1-18
mobility
choosing file in sample sheet 5-35
inconsistent as a result of impurities in the
agent 2-3
inhibited by buffer impurity 2-6
slow, gel troubleshooting 8-14
modem port 5-27
modifying, sample sheet 5-38
See Also editing, sample sheet
Module settings dialog box 3-18
modules 3
ABI PRISM 373 with XL upgrade module
default values B-6
creating user-specific
files 3-17–3-19, 4-20–4-21
importing and exporting 5-67
I-15
Applied Biosystems
opening using small document
icons 3-23, 4-27, 5-40, 5-41
running manually 5-60
mounting gel plates in cassette 2-15–2-17
mRNA
using GeneScan to perform quantitative
gene analysis 1-17
multicomponent analysis, processing
data 1-16
multicomponent matrix 3
choosing 5-35
multiple alignment for DNA 1-18
multiplexing
adding more than one pair of primers
into the same reaction tube 4-6
performing multiple PCRs in separate
reaction tubes 4-7
mutation identification, using Sequence
Navigator Sequence Comparison
software 1-18
one-lane sequencing 1-16
Open command 5-6
opening
Electrophoresis History window 5-57
Run window 5-40–5-41
sample sheet using small document
icons 3-23, 4-27, 5-40, 5-41
optimizing, computer hard disk 7-9
See Also maintenance
organic solvents and glass plates 2-13
output files 5-74
oxygen, affect on gel quality 2-7
P
p53, screening of sequences 1-18
page setup 5-6
specifying parameters about the printout
before printing 5-66
pairwise alignment of DNA 1-18
parameters
setting default parameters
GeneScan mode 4-22–4-24
NaCl (Sodium Chloride) B-10
Sequencing mode 3-19–3-21
naming conventions for DyeSet/Primer
setting temperature control parameters to
files 5-34–5-35
optimize PCR E-10
NaOH (Sodium Hydroxide) B-10
specifying for run 4-26
New command 5-6
Paste command 5-8
noise levels, using the DataUtility software to Pause button 5-46, 5-60
check 1-16, 7-5
pausing to load additional samples 3-43
noisy data (too many peaks), characteristic of Pause command
poor data 4-56
Instrument menu buttons 5-9
non-buffer ions, contaminating buffers 2-6
resuming a pre-run or run 5-48
Norton Utilities
PCR
using to defragment your hard
multiplexing the polymerase chain
drive 7-7–7-10
reaction 4-6
optimizing E-3–E-7
using to optimize the hard disk 7-9
analyzing post-PCR products E-14
avoiding contamination E-12–E-13
performing Hot Start PCR E-8–E-9
old gels, affect on results 2-8
N
O
I-16
Index
March 2001
Applied Biosystems
setting temperature control
parameters E-10
primers
concentration of E-5
storage of 4-63
products
overlapping allele size ranges 4-6
sizing 1-17
templates
concentration of E-6
insufficient concentration of 4-58
troubleshooting 4-58–4-62
PCR Instrument System E-4
See Also thermal cycler
peaks
absence of peaks in results file 4-56
artifacts 4-56
evaluating the shape of peaks 4-54
real time viewing 1-12
removing peaks seen in the Scan
window 3-28
split into two peaks 4-62
too many peaks (noisy data) 4-56
PEG (polyethylene glycol 8000) B-10
photomultipler tube. See PMT
pipetting, correcting errors 4-61
PlateCheck module
template shipped with
system 3-17–3-19, 4-20–4-21
plates. See glass plates
platinum wire 4-64
PMT 3
checking and adjusting gain 5-61–5-64
checking the setting before loading the
sample
using GeneScan mode 4-32–4-34
using Sequencing mode 3-32–3-33
poly (A+) RNA
PCR template concentration E-6
March 2001
Index
polyacrylamide gel. See gel
Polyethylene glycol 8000 B-10
Polymerase Chain Reaction (PCR)
optimizing amplifications E-3–E-14
temperature control parameters E-10
See Also PCR; Hot Start technique
polymerases, with ABI PRISM 373 with XL
upgradeL 1-11
polymerization
affect on gel quality
APS 2-5
buffer impurity 2-6
rate of polymerization 2-6
TEMED 2-5
mechanism of 2-3
too slow (troubleshooting) 8-15
poor resolution
caused by acrylamide impurity 2-4
troubleshooting 8-14
post-PCR analysis E-14
pouring gels
preparing the glass plates 2-22
See Also gels
Power Macintosh computer. See computer
power switch, ABI PRISM 373 with XL
upgradeL 6-17
preferences
Preference file described 5-76
saving 5-14
setting 5-14–5-29
default
general 5-27–5-29
GeneScan
preferences 4-22–4-24
GeneScan Run
defaults 5-26–5-27
GeneScan Sample Sheet
defaults 5-25
preferences 3-19–3-21
I-17
Applied Biosystems
Sequence Run
defaults 5-23–5-24
Sequence Sample Sheet
defaults 5-21–5-22
sequencing analysis
applications 5-21–5-24
Dye Indicators defaults 5-29–5-31
file name preferences 5-18–5-20
folder location
preferences 5-16–5-18
fragment analysis sample sheet 5-25
switching preference windows 5-15
Preferences command 5-11
Preferences Dye Indicators window 5-30
Preferences File Names window 5-19
Preferences Folder Locations window 5-16
Preferences GeneScan Run Defaults
window 5-26
Preferences GeneScan Sample Sheet
Defaults window 5-25
Preferences Sequence Run Defaults
window 5-23
preparing
DNA for loading 3-40
electrophoresis
chamber 3-8–3-15, 4-11–4-18
for Classic configuration 3-9,
4-12–4-13
for Leon configuration 3-10–3-11,
4-11–4-14
for Stretch configuration 3-12–3-15,
4-15–4-18
gel for loading 3-6–3-7
gel solutions
4.0% acrylamide gels
for 48 cm gels 2-25
4.75% gel
24 cm BaseSprinter 2-19
34 cm Full Scan gel 2-19
I-18
Index
acrylamide-urea gel
solutions 2-18–2-19
glass plates
for a 48 cm run 2-22
for gels 2-13–2-14
for GeneScan mode 4-9–4-10
for loading the gel 3-6
Tris Borate EDTA 3-6, 4-9
samples for loading
for electrophoresis 4-42–4-43
GeneScan mode 4-41–4-43
pre-running the gel 4-41
Sequencing mode 3-39–3-44
stock solutions
acrylamide stock solution 2-11
TBE stock solution 2-10–2-11
the gel for loading
in GeneScan mode 4-9–4-10
See Also reagents
PreRun button
starting from the Run window 5-45
to pre-electrophorese gel 3-39, 4-41
Pre-run module
template shipped with
system 3-17–3-19, 4-20–4-21
primer concentration
optimizing PCR reaction E-5–E-6
Primer Express primer design program 1-18
primers
concentration for PCR
optimizing E-5–E-6
extension with commonly used
polymerases 1-11
fluorescent dye-labeled 1-11
kits with dye terminator/dye primer
chemistries 1-11
Primer Express primer design
program 1-18
See Also PCR, primers
March 2001
Applied Biosystems
Print command 5-6
Print One command 5-7
printer port 5-27
printing
automatic printing after
analysis 4-53, 5-67
files 5-66–5-67
matrix values D-10–D-12
page setup 5-66
printing the data automatically using the
analysis program 3-25
sequence files 3-53
using a color printer 1-13
program files. See Data Collection, program
files
protein sequences
alignment using Sequence Navigator 1-18
analyzing with Inherit software 1-17
Public Utilities, using to maintain your hard
disk 7-11
purity
effect of reagent purity on gel
quality 2-3–2-6, 2-9
high purity reagents for gels 2-18
Q
q. s. (Quantity sufficient) B-10
quality
factors affecting gel quality 2-6
maximizing gel resolution 2-9
polymerization rate and gel quality 2-6
quantity of DNA for sequencing 3-5
Quantity sufficient (q.s.) B-10
quick start information 1-8
quitting
Data Collection program 5-68
Quit command 5-6
March 2001
Index
R
radicals 2-3
radioactive labels 1-11
RAM
clearing RAM 7-8
system requirement 7-3
RAPD, using GeneScan to size PCR
products 1-17
raw data
analysis 1-16, 7-5
automatic
analysis 3-50–3-51, 4-53, 5-24, 5-27
color guide for Data Collection program
display of data B-9
displayed 1-13
electropherograms 3-51
in gel files 5-74
special software for data collection and
analysis 7-5–7-6
See also data
reaction mixtures
deoxynucleoside triphosphate, reaction
volume and tube types E-5
enzyme concentration, reaction volume
and tube types E-6–E-7
magnesium ion, reaction volume and tube
types E-5
preparing E-3–E-7
reaction volume and tube
types E-3–E-4
primer concentration, reaction volume
and tube types E-5–E-6
template concentration, reaction volume
and tube types E-6
reaction tubes
correct seating of 4-59
selection of type 4-59
types of E-4
reaction volume E-3–E-4
I-19
Applied Biosystems
table of reaction tube types E-4
read region 6-7, 6-12–6-13, 3
Sequencing scan mode 6-12–6-13
readings under normal conditions 5-51
reagents
affect of purity on gel
quality 2-3–2-6, 2-9, 2-18
diluting with distilled, deionized
water 2-6
preparing stock solutions 2-10–2-11
supplies for preparing gels 2-11–2-12
real-time displays 5-52–5-58
filters and color guide 5-52–5-54
observing data 1-12, C-4, D-4
using the Electrophoresis History
window 5-57–5-58
using the Gel window 3-45, 4-49, 5-56
using the Scan window 3-45, 4-49, 5-55
rebuilding the desktop 7-5
recording sample information 3-21–3-22
red alerts 5-57
red LED 6-19
Redo command 5-8
registering software programs 1-8
removing
beam-stop bar 4-13
non-essential files from hard disk 7-9
the gel from the instrument
in GeneScan mode 4-50–4-51
in Sequencing mode 3-47–3-48
renaturation, consequences of 4-61
reproducibility of the gel
affect of temperature on gel 2-6
affect of vacuum strength and time during
degassing 2-7
compromised by reagent
impurity 2-4–2-6
resin, deionizing. See deionizing resin
I-20
Index
resolution
affected by acrylamide impurity 2-3
maximizing 2-9
poor 8-14
restriction sites E-6
results. See quality
Resume command 5-9, 5-48
resuspending DNA 3-40
See Also DNA
reverse transcriptase
MuLV E-6
rTth DNA Polymerase E-6
Revert command 5-6
rRNA
PCR template concentration E-6
rTth DNA Polymerase E-6
Run file
choosing the length of the run 3-25
creating
for GeneScan mode 4-26–4-29
for Sequencing mode 3-22–3-26
defaults
for fragment analysis 5-25
for sequencing 5-21
example of window 4-27
storing 3-52, 4-65
viewing associated sample sheet or
module 3-23
Run folder
icon for creating Run folder 5-40
output files placed for a run by the
instrument 5-74
Run Log file. See Log file
Run Setup command 5-11
Run window 5-40–5-48
completing
GeneScan Run window for pre-run
or run 5-43–5-45
March 2001
Applied Biosystems
preventing salt concentration in
Sequence window for a pre-run or a
samples 4-6
run 5-41–5-43
sample files 3
opening the window 5-40–5-41
archiving 3-53, 4-66, 5-66
starting a pre-run or run 5-45–5-48
attaching new matrix D-13
from the Instrument menu 5-48
evaluating the quality of the data 4-54
from the Run window 5-45–5-47
characteristics of good data 4-55
runs
characteristics of poor
choosing sample sheet 5-41
data 4-56–4-57
cleaning up after 3-47, 4-50
file format compatible with commercial
clearing random access memory by
programs 1-16
restarting the Macintosh 7-8
output files described 5-75
conditions for starting 5-12
sample sheet 5-32–5-39, 5-74
example of run window 4-27
adding default values 5-34
importing and exporting 5-68
applying the same parameter to all fields
preparing software 5-12
in a column 5-38
problems that might occur during a
choosing for run 5-41
run 8-13
copying information 5-38
readings under normal conditions 5-51
creating
resetting global serial number (for file
GeneScan sample sheet (using Data
names) 5-27
Collection
Run Log described 5-74
software) 5-36–5-37
run time in modules B-6
in GeneScan mode 4-24–4-26
setting
in Sequence mode 3-21–3-22
defaults for sequence runs 5-23
Sequence sample sheet (using Data
parameters 4-26
Collection
starting, stopping, pausing 5-48
software) 5-34–5-35
status during run 3-45
defaults
for fragment analysis 5-25
for sequencing 5-21
.seq file 5-75
DyeSet/Primer file naming
safety warnings 1-6
conventions 5-34–5-35
acrylamide 2-9, 2-18
exporting 5-67
acrylamide and bis-acrylamide 2-4
fixing truncated entries 3-24
bis-acrylamide 2-9, 2-18
importing and exporting sample sheet
laser hazard 3-28, 4-31
information 5-33
TEMED 2-5
importing from text files 5-68
urea 2-6, 2-18
information on electropherogram 3-51
salt concentration
modifying an existing sample sheet 5-38
effect on PCR 4-59
S
March 2001
Index
I-21
Applied Biosystems
moving
around sample sheet 5-36
between fields in the sample
sheet 5-38
information to a different lane 5-39
separate for sequence and fragment
analysis 3-21, 5-32
viewing
from Run window 3-23, 5-40
information associated with the current run 5-58
Sample Sheet command 5-11
samples
automatic analysis by
GeneScan Analysis program 5-27
Sequencing Analysis program 5-24
automatic analysis of
sample 3-50–3-51, 4-53
evaluating the contamination of unknown
samples 4-54
identifying the sample 5-32
loading
GeneScan mode 4-43–4-47
Sequencing mode 3-41–3-44
preparing
for electrophoresis 4-42–4-43
for GeneScan mode 4-41
samples before instrument setup 3-5
samples for GeneScan mode 4-5
pre-running the gel 3-39
preventing high salt concentration 4-6
recording information for analysis 5-32
resolution of samples decreased 4-63
specifying lane for run 4-24, 4-26, 5-32
See Also sample sheet, creating
saving 5-6
automatically created files, ensuring disk
space 3-27, 3-42, 4-30, 4-45, 4-46
I-22
Index
backup copy of the file you are working
with 5-65
files 5-65
created during a
run 3-52–3-53, 4-65–4-66
other files 3-53
matrix in ABI folder D-3
module settings 3-18–3-19
preferences 5-14
sample files 5-66
scaling
Electrophoresis History window 5-58
processing data 1-16
Scan window 5-55, 5-62
Scan command 5-11
scan numbers
electrophoresis history window 5-57
scan point 1
Scan window
checking the plates 4-31
setting scale 5-55, 5-62
viewing data 1-13
in real-time 3-45, 4-49, 5-55
scanning
gels 4-30–4-31
plates 3-27–3-38, 4-30–4-31
using the Scan window 4-31
scanning and detection system 6-11–6-16
filter wheel and data display 6-13–6-15
instrument and matrix files 6-15–6-16
read region 6-12–6-13
SDS (Sodium dodecyl sulfate) B-11
searching biological databases using
GeneAssist 1-18
secondary structure
of RNA templates E-6
Select All command 5-8
separation distance 3
separation system 6-10
March 2001
Applied Biosystems
sequence files, output files described 5-75
Sequence Navigator software 1-18
Sequence Run defaults 3-20
Sequence Sample Sheet
creating the sample sheet 3-21–3-26
defaults 3-19
Sequence window
completing for pre-run or run 5-41–5-43
sequences 3
sequencing
data interpretation 3-50–3-51
detection described 2-9
effect of reagent purity on gel
quality 2-3–2-6
GelDocII and DataUtility programs 1-16
one-lane approach 1-16
optimal quantities of DNA 3-5
sample sheet
selecting sample sheet 5-32
setting defaults for sample sheets,
runs 5-21
using separate sample sheet 3-21
See Also sample sheet
set custom parameters before
run 4-8, 4-19
setting up for automatic analysis 3-15
table of run times and filter sets B-7
template shipped with
system 3-17–3-19, 4-20–4-21
using a setting other than default for
automatic analysis 3-5
using the ABI PRISM 373 with XL
upgradeL 1-10
using the ABI PRISM 373 with XL upgrade
1-12
Sequencing Analysis applications
setting defaults 5-21–5-24
Sequencing Analysis program 5-75
GelDocII and Data Utility programs 1-16
March 2001
Index
using after data collection 5-17
sequencing Instrument file
chemistries and the Instrument file C-3
See Also matrix files
sequencing reactions 3
serial number, reset for file naming 5-27
Set Scale command 5-8
setting
defaults for fragment analysis 5-25
general preferences 5-27
temperature control parameters for
optimizing PCR E-10
the electrophoresis chamber for
sequencing applications 3-12
setting up the instrument 3-6–3-38
checking
the plates 3-27–3-38, 4-30–4-40
the PMT setting 3-32–3-33,
4-32–4-34
completing the setup 3-35–3-38
GeneScan mode 4-9–4-40
completing instrument
setup 4-36–4-40
for Classic
configuration 4-36–4-37
for Leon
configuration 4-37–4-39
for Stretch
configuration 4-39–4-40
preparing the electrophoresis
chamber 4-11–4-18
for Classic
configuration 4-12–4-13
for Leon
configuration 4-13–4-14
for Stretch
configuration 4-11–4-18
preparing the gel for
loading 4-9–4-10
I-23
Applied Biosystems
inserting the Sharks-tooth comb 3-34
instrument connection to Macintosh 5-27
preparing the electrophoresis
chamber 3-8–3-15
for Classic configuration 3-9
for Leon configuration 3-10–3-11
for Stretch configuration 3-12–3-15
preparing the gel for loading 3-6–3-7
setting up the software
analysis software 3-16
creating the Run file 3-22–3-26
choosing the length of the run 3-25
GeneScan mode 4-19–4-29
creating a Run file 4-26–4-29
creating the sample sheet 4-24–4-26
creating user-specific module
files 4-20–4-21
setting default parameters 4-22–4-24
starting the Data Collection
software 4-20–4-29
the analysis software 4-19
Sequencing mode 3-16–3-26
creating the sample sheet 3-21–3-26
creating user-specific module
files 3-17–3-19
setting default parameters 3-19–3-21
starting the Data Collection
software 3-17
settings 3
changing the instrument settings in
real-time 5-59–5-60
for Sequencing Analysis, setting must be
ABI folder 5-17
page for printing 5-66
Sharks-tooth comb 6-8, 3
casting the gel 2-20
channel numbers at the center of each
lane 3-29–3-31
inserting the comb 3-34
I-24
Index
GeneScan mode 4-34–4-35
loading many samples 3-41
Show Clipboard command 5-8
signal
intensity of 4-62
strength for best results 3-40
See Also peaks
Silating plates. See Bind-Silane
SilverLining
using to maintain your hard disk 7-11
size standard
setting default 5-25
sizing and quantitating DNA
fragments 1-9, 7-5
skipping lanes
in GeneScan mode 4-32
in Sequencing mode 3-29–3-31
skipping multiple lanes loading
gel 3-42, 4-44, 4-45
slow mobility, troubleshooting 8-14
slow polymerization, troubleshooting 8-15
small document icons on the Run
window 3-23, 4-27, 5-40, 5-41
Sodium dodecyl sulfate (SDS) B-11
software
adding software programs 7-10
AutoAssembler™ 1-17, 1-18
backing up 7-8
copying image file to firmware 6-17
data collection program files 5-73–5-74
DataUtility
bundled with Sequencing Analysis
program 1-16
using to make and copy matrices 7-5
Factura software 1-17
for data analysis 1-16–1-18
GelDocII 1-16
Genotyper™ 1-18
installing 7-4–7-5
March 2001
Applied Biosystems
performing raw data collection and data
analysis 7-5–7-6
preparing for run 5-12
provided with instrument 7-5
registering 1-8
Sample file format compatibility 1-16
Sequence Navigator 1-18
troubleshooting 8-16
See Also setting up the software
spacers
compatibility with combs and casting
comb 3-34
See gel spacers
special text in the manual 1-5
spectral overlap
corrected with matrix D-3
spills, cleaning up E-13
split peaks. See peaks
spreadsheets
Excel, exporting the sample sheet
to® 5-74
exporting files for 5-67
for use with Genotyper data 1-18
square-tooth comb 6-9, 3
applying Bind-Silane compound 2-14
casting the gel 2-20
loading lanes consecutively 4-44
using to load lanes
consecutively 3-42, 4-45
See Also combs
standards. See matrix files
Start Plate Check command 5-9
Start Run command 5-9, 5-48
starting
conditions for starting runs 5-12
pre-run or run from the Run
window 5-45–5-48
from the Instrument menu 5-48
from the Run window 5-45–5-47
March 2001
Index
quick start information 1-8
Start commands 5-9
status
lights on instrument 6-17, 6-19
log file 4-48
table of status light states 6-20
viewing instrument status
in GeneScan mode 4-48–4-49
in Sequencing mode 3-45–3-46
using the Status window 5-50–5-51
See also translation interface processor,
status and control; viewing
Status command 5-11
Status window, using to view instrument
status 5-50–5-51
stock solutions
10X TBE buffer 2-10–2-11
40% acrylamide 2-11
preparing fresh acrylamide, bis
acrylamide, urea 2-6
purchasing powders and
diluting 2-4, 2-10
stopping a pre-run or run 5-46
storing
acrylamide 2-4
APS 2-5
bis-acrylamide 2-4
software files 3-52, 4-65–4-66
TEMED 2-5
See Also saving
STR, using GeneScan to size PCR
products 1-17
Stretch, Model 373 DNA Sequencer
configuration 1-4, 3
completing instrument
setup 3-37–3-38, 4-39–4-40
model specific notation 3-8, 4-11
preparing the electrophoresis
chamber 3-12–3-15, 4-15–4-18
I-25
Applied Biosystems
sulfate radical, yielded from polymerization
mechanism 2-3
supplies for gels 2-11–2-12
swirls in gel, troubleshooting 8-15
system folder
files located in 5-76
temporary files created in 5-76
system requirements 7-3
T
T7 DNA polymerase
primer extension 1-11
tab-delimited text
format 5-67
importing or exporting 5-33
tables
abbreviations B-10–B-11
channel numbers in lanes 3-29–3-31
colors of bases in real-time
displays 5-53, B-9, C-4
DNA sequencing resuspension and
loading volumes 3-40
dye sets and filters sets by chemistry B-9
dyes and filter set color displays D-5
files in System folder 5-76
GeneScan mode :sample, cocktail, and
loading volumes 4-43
how changing temperature control
parameters affects the PCR E-11
input files in the Data Collection
program 5-73–5-74
instrument menu commands 5-9
IUB codes and their complements 2
matrix standard dyes and part
numbers D-6
module file default
parameters 3-17, 4-20
module parameters B-6
I-26
Index
optimal quantity of template DNA for
sequencing 3-5
output files in the Data Collection
program 5-74
problems that might occur during a
run 8-13
reaction tube types for PCR instrument
systems E-4
relationships between filters, sequencing
dyes, and data display 6-14
run readings under normal
conditions 5-51
status light states 6-20
troubleshooting gels 8-14–8-15
tared 3
TBE B-11
buffer 4-38, 4-64
preparing stock solution 2-10–2-11
TE, 10 mM Tris-Cl (pH 8.0), 1 m M EDTA
(pH 8.0) B-11
technical support 1-22–1-28
registering software for 1-8
technical updates to user’s manual 1-7
TEMED B-11
adding to cast the gel 2-20
affect of
changing concentration 2-7
impurity 2-5
chemical hazard 2-11
safety warning 2-5
storage 2-5
use in mechanism of polymerization 2-3
temperature
affect on gel quality 2-6–2-7
control parameters, effect on PCR
performance E-10
gel status 5-57
status of gel 1-12
March 2001
Applied Biosystems
template
PCR concentration E-6
using module file
templates 3-17–3-19, 4-20–4-21
temporary files 5-76
tetramethylethylenediamine. See TEMED
text files 4
exporting ABI PRISM 373 with XL
upgradeL data to 5-67
thermal control plate. See heat transfer plate
thermal cycling
cleaning sample block E-13
setting parameters for 4-58
thermal equilibration E-11
total cellular RNA
PCR template concentration E-6
total reset
procedure for clearing memory 8-10
Translation Information Processor
definition 4
status and control 6-120
Translation Interface Processor
ABI PRISM 373 with XL upgrade DNA
Sequencer upgrade 1-9
status and control 6-17–6-20
when starting the Data Collection
program 3-17, 4-20
Translation Processor
diagram showing flow of data 5-72
Tri hydrochloride (Tris-CI) B-11
Tris Borate EDTA
preparing the glass plates 3-6, 4-9
Tris contaminants 2-6
Tris-Cl (Tris hydrochloride) B-11
tRNA, PCR template concentration E-6
troubleshooting 8-8–8-16
clearing the memory 8-9–8-12
gel troubleshooting 8-14–8-15
hardware/firmware 8-9–8-12
March 2001
Index
PCR and electrophoresis conditions table
for GeneScan mode 4-58
problems during a run 8-13
total reset of the instrument 8-8
troubleshooting software 8-16
tube types E-3–E-4
two-dimensional array 5-32
U
Undo command 5-8
UNIX
client-server option for the
AutoAssembler Sequence Assembly
Software 1-18
using Inherit Sequence Analysis
System 1-17
upper buffer chamber replacing. See
individual instrument configuration
urea
affect of impurity 2-6
chemical hazard 2-11
contaminants, found in 2-6
safety warning 2-6, 2-18
solution for gel 2-18–2-19
user attention words 1-5
User Bulletins, updated to the manual 1-7
user’s manual
how to use this manual 1-3–1-5
updates 1-7
user attention words 1-5
V
verifying lane tracking D-10
viewing
associated sample sheet or module 5-40
data collection status windows 3-45, 4-48
instrument status
data in real-time 5-52–5-58
during and after a run 5-50–5-58
I-27
Applied Biosystems
filters and color guide 5-52–5-54
using the Electrophoresis History window 5-57–5-58
using the Gel window 5-56
using the Scan window 5-55
Run file for the current run 5-58
sample sheet associated with the
run 5-58
using
the Log file 5-51–5-52
the Status window 5-50–5-51
real-time data windows 3-45, 4-49
See Also Status window; Electrophoresis
History window; The Log file; Scan
window; Gel window
viral RNA, PCR template concentration E-6
virus protection, during installation 7-4
VNTR, sizing with GeneScan® software 1-17
voltage
actual voltage and current readings
during a run 5-50
in modules B-6
W
using Bind-Silane compound to eliminate
deformed wells 2-14
using formamide in empty
wells 3-42, 4-44, 4-45
well-to-read length. See WTR
Window menu 5-10–5-11
windows
changing size of 5-10, 5-49
closing 5-10, 5-49, 5-68
default settings 5-14
moving on screen 5-10, 5-49
switching preferences windows 5-15
See Also individual window names
word processing
exporting files for 5-67
sequence files
exporting 5-75
using to open sequence files 3-53
WTR (well-to-read)
definition 4
setting default for run file
GeneScan Analysis program 5-27
Sequencing Analysis program 5-24
table of abbreviations B-11
warnings
acrylamide 2-4, 2-9, 2-18
X-GAL (Microgram) B-10
bis-acrylamide 2-4, 2-9, 2-18
XL Scan mode 6-12
TEMED 2-5
urea 2-6, 2-18
warranty A-1–A-2
washing plates 2-13, 3-48
See Also cleaning
watts, in modules B-6
See Also voltage
wells
depressions in the well surface 2-27
flushing before loading 4-41
loading alternate wells with Shark’s-tooth
comb 3-41
X
I-28
Index
March 2001
Headquarters
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Phone: +1 650.638.5800
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Fax: +1 650.638.5884
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Applera Corporation is committed to providing the
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Printed in the USA, 03/2001
Part Number 904258C
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