Applied Biosystems 3730 xl DNA Analyzer User Guide
The Applied Biosystems 3730/3730xl DNA Analyzers are used for sequencing and fragment analysis. They are capable of analyzing 96 samples simultaneously with a throughput of up to 96 samples per hour. The 3730/3730xl DNA Analyzers are compatible with a variety of Applied Biosystems reagents and software.
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User Guide
Applied Biosystems
3730/3730xl
DNA Analyzers
For use with Data Collection Software v2.0
Preparing the
Instrument
Performing
Spatial
Calibration
Performing
Spectral
Calibration
Setting Up the Plate
Running the
Instrument
Shutting
Down the
Instrument
Performing
Instrument
Maintenance
© Copyright 2003, Applied Biosystems. All rights reserved.
For Research Use Only. Not for use in diagnostic procedures.
Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that may appear in this document. This document is believed to be complete and accurate at the time of publication. In no event shall Applied Biosystems be liable for incidental, special, multiple, or consequential damages in connection with or arising from the use of this document.
NOTICE TO PURCHASER:
This instrument, Serial No. __________, is Authorized for use in DNA sequencing and fragment analysis. This authorization is included in the purchase price of this instrument and corresponds to the up-front fee component of a license under process claims of U.S. Patent Nos. 5,821,058 and 5,332,666 and under all process claims for DNA sequence and fragment analysis of
U.S. patents now or hereafter owned or licensable by Applied Biosystems for which an Authorization is required, and under corresponding process claims in foreign counterparts of the foregoing for which an Authorization is required. The running royalty component of licenses may be purchased from Applied Biosystems or obtained by using Authorized reagents purchased from Authorized suppliers in accordance with the label rights accompanying such reagents. Purchase of this instrument does not itself convey to the purchaser a complete license or right to perform the above processes. This instrument is also licensed under U.S. Patent No. 5,171,534 and apparatus and system claims in foreign counterparts thereof. No rights are granted expressly, by implication or by estoppel under composition claims or under other process or system claims owned or licensable by Applied Biosystems. For more information regarding licenses, please contact the Director of Licensing at
Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.
NOTICE TO PURCHASER:
The purchase price of this [Capillary Sequencing Instrument Name] includes a grant of a limited, non-transferable license under U.S. Patent No. 5,567,292 and method claims of its foreign counterparts, and under U.S. Patent No. 6,358,385 and element claims of its foreign counterparts, to use this particular instrument for electrophoresis methods employing fluorescence as a means of detection. No other licenses or rights are hereby conveyed either expressly, by implication, or estoppel including, but not limited to, any claims to a composition.
The Applied Biosystems 3730 and 3730xl DNA Analyzer includes patented technology licensed from Hitachi, Ltd. as part of a strategic partnership between Applied Biosystems and Hitachi, Ltd., as well as patented technology of Applied Biosystems.
TRADEMARKS:
ABI PRISM, Applied Biosystems, BigDye, and MicroAmp are registered trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries.
ABI, GeneMapper, GeneScan, Hi-Di, POP, and POP-7, are trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries.
Microsoft, Windows, and Windows NT are registered trademarks of the Microsoft Corporation in the United States and/or other countries.
Oracle is a registered trademark of the Oracle Corporation.
pGEM is a registered trademark of Promega Corporation.
All other trademarks are the sole property of their respective owners.
4347118 Rev. B
12/2003
3730/3730xl Workflow
Chapter
Chapter
Chapter
Chapter
Chapter
1
2
3
4
5
Preparing the
Instrument
Start the 3730/3730xl
Data Collection
Software
Install the capillary array
Add and replace the polymer
Performing
Spatial Calibration
Perform spatial calibration
Evaluate the calibration data
Performing
Spectral Calibration for Sequencing and
Fragment Analysis
Setting Up the Software for
DNA Sequencing
Prepare the spectral calibration chemistry
Create required settings for automated sequencing analysis
Create a spectral instrument protocol
Create and complete a sequencing analysis plate record
Setting Up the Software for
Fragment Analysis
Create required settings for automated fragment analysis
Create and complete
a GeneMapper plate record
Perform spectral calibration
Fill down special
Prepare buffer and fill the reservoirs
Evaluate the spectral calibration data
Chapter
6
Running the
Instrument
Work with plate assemblies
Place plate assemblies into the instrument
Schedule
a run
Run the
instrument
Control the run
Work with data in the Run History view
View analyzed data
Chapter
Chapter
7
8
Performing
Maintenance
Audit Trails and
Access Control
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Clean the pump block and lower polymer block
Enable the access control and audit features
Flush and fill the water trap
Perform a short-term shutdown
Store a capillary array
Archive data
Defragment the computer hard drives
Delete records from the database
Start the
AB Navigator
Configure
the audit map
Audit
View the audit history
Export/import user settings, applications, group profiles
Set up password policies
Access Control
Create a new user
Create a new profile
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Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Contents
3730/3730xl Workflow
Preface
Safety
Chapter 1
Preparing the Instrument 1
Chapter 2
Performing Spatial Calibration 19
Chapter 3
Performing Spectral Calibration For Sequencing and
Fragment Analysis 29
Examples of Passing Sequencing Spectral Calibrations . . . . . . . . . . . . . . . . . . . . . . .49
Example of a Passing Fragment Analysis Spectral Calibration . . . . . . . . . . . . . . . . . .51
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
v iii
ix
xi
Chapter 4
Setting Up the Software for DNA Sequencing 57
Creating Required Settings for Automated Sequencing Analysis . . . . . . . . . . . . . . . . 62
Creating and Completing a Sequencing Analysis Plate Record . . . . . . . . . . . . . . . . . . 79
Chapter 5
Setting Up the Software for Fragment Analysis 87
3730/3730xl Data Collection and GeneMapper Software . . . . . . . . . . . . . . . . . . . . . . 88
Creating Required Settings for Automated Fragment Analysis . . . . . . . . . . . . . . . . . . 95
Creating and Completing a GeneMapper Plate Record . . . . . . . . . . . . . . . . . . . . . . . 108
Chapter 6
Running the Instrument 115
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Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Chapter 7
Performing Maintenance 153
Guidelines for Pump Block and Lower Polymer Block Cleaning . . . . . . . . . . . . . . . .157
Working With Drives for Database and Sample Data Storage . . . . . . . . . . . . . . . . . .174
Chapter 8
Audit Trails and Access Control 181
Exporting User Settings, Applications, and Group Profiles . . . . . . . . . . . . . . . . . . . .195
Importing User Settings, Applications, and Group Profiles . . . . . . . . . . . . . . . . . . . .196
Appendix A Parts List
209
Appendix B G5, G5-RCT, Any4Dye, and Any5Dye Dye Sets
211
Creating a Spectral Calibration for the Any4Dye or Any5Dye Dye Sets . . . . . . . . . . .213
Regular Runs Using Any4Dye or Any5Dye Dye Sets . . . . . . . . . . . . . . . . . . . . . . . . .217
Index 221
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
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Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Preface
How to Use This Guide
Purpose of This
Guide
This guide is written for the training of principle investigators and laboratory staff who operate and maintain the Applied Biosystems 3730/3730xl DNA Analyzers.
Assumptions
This guide assumes the following background:
• Familiarity with the Microsoft
®
Windows
®
2000 operating system.
• Knowledge of techniques for handling and preparing DNA samples for sequencing.
• A general understanding of hard drives and data storage, file transfers, and copying and pasting.
Text Conventions
This guide uses the following conventions:
• Bold indicates user action. For example:
Type 0, then press Enter for each of the remaining fields.
• Italic text indicates new or important words and is also used for emphasis. For example:
Before analyzing, always prepare fresh matrix.
• A right arrow bracket (>) separates successive commands you select from a dropdown or shortcut menu. For example:
Select File > Open > Spot Set.
Right-click the sample row, then select View Filter > View All Runs.
User Attention
Words
Two user attention words appear in Applied Biosystems user documentation. Each word implies a particular level of observation or action as described below:
Note:
Provides information that may be of interest or help but is not critical to the use of the product.
IMPORTANT!
Provides information that is necessary for proper instrument operation, accurate chemistry kit use, or safe use of a chemical.
Examples of the user attention words appear below:
Note:
The size of the column affects the run time.
Note:
The Calibrate function is also available in the Control Console.
IMPORTANT!
To verify your client connection to the database, you need a valid Oracle user ID and password.
IMPORTANT!
96-well plate.
You must create a separate Sample Entry Spreadsheet for each
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
ix
Preface
Send Us Your Comments
Safety Alert
Words
Safety alert words also appear in user documentation. For more information, see
Send Us Your Comments
Applied Biosystems welcomes your comments and suggestions for improving its user documents. You can e-mail your comments to:
x
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Safety
Safety Alert Words
Four safety alert words appear in Applied Biosystems user documentation. Each word implies a particular level of observation or action, as described below:
IMPORTANT!
Indicates information that is necessary for proper instrument operation, accurate chemistry kit use, or safe use of a chemical.
Indicates a potentially hazardous situation that, if not avoided, may result in minor or moderate injury. It may also be used to alert against unsafe practices, damage to an instrument, or loss of data.
Indicates a potentially hazardous situation that, if not avoided, could result in death or serious injury.
Indicates an imminently hazardous situation that, if not avoided, will result in death or serious injury.
Sources of Safety Information
For System Operators
Operational safety information for the Applied Biosystems 3730/3730xl DNA Analyzer is provided in the following documents, which are included with each system.
Material Safety Data Sheets (MSDSs)
MSDSs provide information you need to store, handle, transport, and dispose of chemicals safely.
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Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Safety
Symbols on Instruments
Symbols on Instruments
Electrical Symbols
The following electrical symbols may be displayed on Applied Biosystems instruments.
Symbol Description
Indicates the On position of the main power switch.
Indicates the Off position of the main power switch.
Indicates the On/Off position of a push-push main power switch.
Indicates a terminal that can receive or supply alternating current or voltage.
Symbol Description
Indicates a terminal that may be connected to the signal ground reference of another instrument.
This is not a protected ground terminal.
Indicates a protective grounding terminal that must be connected to earth ground before any other electrical connections are made to the instrument.
Indicates a terminal that can receive or supply alternating or direct current or voltage.
Safety Symbols
The following safety symbols may be displayed on Applied Biosystems instruments.
Each symbol may appear by itself or in combination with text that explains the relevant hazard (see
“Safety Labels on Instruments” on page xiii ). These safety symbols may also
appear next to DANGERS, WARNINGS, and CAUTIONS that occur in the text of this and other product-support documents.
Symbol Description
Indicates that you should consult the manual for further information and to proceed with appropriate caution.
Indicates the presence of an electrical shock hazard and to proceed with appropriate caution.
Indicates the presence of a hot surface or other hightemperature hazard and to proceed with appropriate caution.
Symbol Description
Indicates the presence of a laser inside the instrument and to proceed with appropriate caution.
Indicates the presence of moving parts and to proceed with appropriate caution.
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Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Safety
Safety Symbols
Safety Labels on Instruments
The following CAUTION, WARNING, and DANGER statements may be displayed on
Applied Biosystems instruments in combination with the safety symbols described in the preceding section.
English
CAUTION Hazardous chemicals. Read the
Material Safety Data Sheets (MSDSs) before handling.
CAUTION Hazardous waste. Read the waste profile (if any) in the site preparation guide for this instrument before handling or disposal.
CAUTION Hazardous waste. Refer to
MSDS(s) and local regulations for handling and disposal.
WARNING Hot lamp.
WARNING Hot. Replace lamp with an
Applied Biosystems lamp.
CAUTION Hot surface.
DANGER High voltage.
WARNING To reduce the chance of electrical shock, do not remove covers that require tool access. No user-serviceable parts are inside.
Refer servicing to Applied Biosystems qualified service personnel.
DANGER Class II laser radiation present when open and interlock defeated. Do not stare directly into the beam
DANGER Class II laser radiation present when open. Do not stare directly into the beam.
CAUTION Moving parts.
Francais
ATTENTION Produits chimiques dangeureux.
Lire les fiches techniques de sûreté de matériels avant la manipulation des produits.
ATTENTION Déchets dangereux. Lire les renseignements sur les déchets avant de les manipuler ou de les éliminer.
ATTENTION Déchets dangereux. Lire les fiches techniques de sûreté de matériels et la régulation locale associées à la manipulation et l'élimination des déchets.
AVERTISSEMENT Lampe brûlante.
AVERTISSEMENT Composants brûlants.
Remplacer la lampe par une lampe
Applied Biosystems.
ATTENTION Surface brûlante.
DANGER Haute tension.
AVERTISSEMENT Pour éviter les risques d'électrocution, ne pas retirer les capots dont l'ouverture nécessite l'utilisation d'outils.
L’instrument ne contient aucune pièce réparable par l’utilisateur. Toute intervention doit être effectuée par le personnel de service qualifié de Applied Biosystems.
DANGER de Class II rayonnement laser en cas d'ouverture et d'une neutralisation des dispositifs de securite. Eviter toute exposition directe avec le faisceau.
DANGER de Class II rayonnement laser en cas d'ouverture. Eviter toute exposition directe avec le faisceau.
ATTENTION Parties mobiles.
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
xiii
Safety
General Instrument Safety
General Instrument Safety
PHYSICAL INJURY HAZARD. Use this product only as specified in this document.
Using this instrument in a manner not specified by Applied Biosystems may result in personal injury or damage to the instrument.
Moving and Lifting the Instrument
PHYSICAL INJURY HAZARD. The instrument is to be moved and positioned only by the personnel or vendor specified in the applicable site preparation guide. If you decide to lift or move the instrument after it has been installed, do not attempt to lift or move the instrument without the assistance of others, the use of appropriate moving equipment, and proper lifting techniques. Improper lifting can cause painful and permanent back injury. Depending on the weight, moving or lifting an instrument may require two or more persons.
Moving and Lifting Stand-Alone Computers and Monitors
Do not attempt to lift or move the computer or the monitor without the assistance of others. Depending on the weight of the computer and/or the monitor, moving them may require two or more people.
Before lifting the computer and/or the monitor:
• Make sure that you have a secure, comfortable grip on the computer or the monitor when lifting.
• Make sure that the path from where the object is to where it is being moved is clear of obstructions.
• Do not lift an object and twist your torso at the same time.
• Keep your spine in a good neutral position while lifting with your legs.
• Participants should coordinate lift and move intentions with each other before actually lifting and carrying.
• Instead of lifting the object from the packing box, carefully tilt the box on its side and hold it stationary while someone slides the contents out of the box.
Operating the Instrument
Ensure that anyone who operates the instrument has:
• Received instructions in both general safety practices for laboratories and specific safety practices for the instrument.
• Read and understood all applicable Material Safety Data Sheets (MSDSs).
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Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Safety
Chemical Hazard Warnings
Chemical Safety
Chemical Hazard Warnings
CHEMICAL HAZARD. Before handling any chemicals, refer to the Material Safety
Data Sheet (MSDS) provided by the manufacturer, and observe all relevant precautions.
CHEMICAL HAZARD. All chemicals in the instrument, including liquid in the lines, are potentially hazardous. Always determine what chemicals have been used in the instrument before changing reagents or instrument components. Wear appropriate eyewear, protective clothing, and gloves when working on the instrument.
CHEMICAL HAZARD. Four-liter reagent and waste bottles can crack and leak. Each
4-liter bottle should be secured in a low-density polyethylene safety container with the cover fastened and the handles locked in the upright position. Wear appropriate eyewear, clothing, and gloves when handling reagent and waste bottles.
MSDSs
Chemical manufacturers supply current Material Safety Data Sheets (MSDSs) with shipments of hazardous chemicals to new customers. They also provide MSDSs with the first shipment of a hazardous chemical to a customer after an MSDS has been updated.
MSDSs provide the safety information you need to store, handle, transport, and dispose of the chemicals safely.
Each time you receive a new MSDS packaged with a hazardous chemical, be sure to replace the appropriate MSDS in your files.
Obtaining MSDSs
You can obtain from Applied Biosystems the MSDS for any chemical supplied by
Applied Biosystems. This service is free and available 24 hours a day.
To obtain MSDSs:
1.
Go to https://docs.appliedbiosystems.com/msdssearch.html
2.
In the Search field, type in the chemical name, part number, or other information that appears in the MSDS of interest. select the language of your choice, then click
Search.
3.
Find the document of interest, right-click the document title, then select any of the following:
• Open – To view the document
• Print Target – To print the document
• Save Target As – To download a PDF version of the document to a destination that you choose
4.
To have a copy of a document sent by fax or e-mail, select Fax or E-mail to the left of the document title in the Search Results page, then click RETRIEVE
DOCUMENTS at the end of the document list.
5.
After you enter the required information, click View/Deliver Selected Documents
Now.
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
xv
Safety
Chemical Safety
Chemical Safety Guidelines
• Read and understand the MSDSs provided by the chemical manufacturer before you store, handle, or work with any chemicals or hazardous materials.
• Minimize contact with chemicals. When handling chemicals, wear appropriate personal protective equipment such as safety glasses, gloves, and protective clothing. For additional safety guidelines, consult the MSDS.
• Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with adequate ventilation (for example, a fume hood). For additional safety guidelines, consult the MSDS.
• Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the cleanup procedures recommended in the MSDS.
• Comply with all local, state/provincial, and/or national laws and regulations related to chemical storage, handling, and disposal.
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Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Safety
Chemical Waste Safety Guidelines
Chemical Waste Safety
CHEMICAL WASTE HAZARD. Some wastes produced by the operation of the instrument or system are potentially hazardous and can cause injury, illness, or death.
Chemical Waste Safety Guidelines
• Read and understand the MSDSs for the chemicals in a waste container before you store, handle, or dispose of chemical waste.
• Provide primary and secondary waste containers
• Minimize contact with and inhalation of chemical waste. When handling chemicals, wear appropriate protective equipment such as safety glasses, gloves, and protective clothing.
• Handle chemical wastes in a fume hood.
• After you empty a chemical waste container, seal it with the cap provided.
• Dispose of the contents of a waste container in accordance with good laboratory practices and local, state/provincial, and/or national environmental and health regulations.
Waste Profiles
A waste profile for the 3730/3730xl DNA analyzer is provided in the 3730/3730xl DNA
Analyzer Site Preparation Guide.
Waste profiles show the percentage compositions of the reagents in the waste stream generated during installation and during a typical user application, even though the typical application may not be used in your laboratory.
The waste profiles help you plan for the handling and disposal of waste generated by operation of the instrument. Read the waste profiles and all applicable MSDSs before handling or disposing of chemical waste.
Waste Disposal
If potentially hazardous waste is generated when you operate the instrument, you must:
• Characterize (by analysis if necessary) the waste generated by the particular applications, reagents, and substrates used in your laboratory.
• Ensure the health and safety of all personnel in your laboratory.
• Ensure that the instrument waste is stored, transferred, transported, and disposed of according to all local, state/provincial, and/or national regulations.
IMPORTANT!
Radioactive or biohazardous materials may require special handling, and disposal limitations may apply.
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
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Safety
Electrical Safety
Electrical Safety
Shock Hazards
ELECTRICAL SHOCK HAZARD. Severe electrical shock can result from operating the 3730/3730xl DNA analyzer without its instrument panels in place. Do not remove instrument panels. High-voltage contacts are exposed when instrument panels are removed from the instrument.
Fuses
ELECTRICAL SHOCK HAZARD. Improper fuses or high-voltage supply can damage the instrument wiring system and cause a fire. Before turning on the 3730/3730xl
DNA analyzer, verify that the fuses are properly installed and that the instrument voltage matches the power supply in your laboratory.
FIRE HAZARD. For continued protection against the risk of fire, replace fuses only with fuses of the type and rating specified for the instrument.
Power Supply
ELECTRICAL HAZARD. Grounding circuit continuity is vital for the safe operation of equipment. Never operate equipment with the grounding conductor disconnected.
ELECTRICAL HAZARD. Use properly configured and approved line cords for the voltage supply in your facility.
ELECTRICAL HAZARD. Plug the system into a properly grounded receptacle with adequate current capacity.
Overvoltage Rating
The 3730/3730xl DNA Analyzer system has an installation (overvoltage) category of II, and is classified as portable equipment
Physical Hazard Safety
Moving Parts
PHYSICAL INJURY HAZARD.
Moving parts can crush and cut. Keep hands clear of moving parts while operating the 3730/3730xl DNA Analyzer. Disconnect power before servicing the 3730/3730xl DNA Analyzer.
PHYSICAL INJURY HAZARD.
Do not operate the 3730/3730xl DNA Analyzer without the arm shield in place. Keep hands out of the deck area when the 3730/3730xl instrument autosamplers are moving.
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Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Safety
Solvents and Pressurized Fluids
Solvents and Pressurized Fluids
PHYSICAL INJURY HAZARD. Always wear eye protection when working with solvents or any pressurized fluids.
PHYSICAL INJURY HAZARD. To avoid hazards associated with high-pressure fluids in polymeric tubing:
• Be aware that Radel
®
tubing is a polymeric material. Use caution when working with any polymer tubing that is under pressure.
• Always wear eye protection when in proximity to pressurized polymer tubing.
• Extinguish all nearby flames if you use flammable solvents.
• Do not use Radel
®
tubing that has been severely stressed or kinked.
• Do not use Radel
®
tubing with tetrahydrofuran or concentrated nitric and sulfuric acids.
• Be aware that methylene chloride and dimethyl sulfoxide cause Radel
® and greatly reduce the rupture pressure of the tubing.
tubing to swell
• Be aware that high solvent flow rates (~40 mL/min) may cause a static charge to build up on the surface of the tubing. Electrical sparks may result.
Biological Hazard Safety
BIOHAZARD. Biological samples such as tissues, body fluids, and blood of humans and other animals have the potential to transmit infectious diseases. Read and follow the guidelines published in:
• U.S. Department of Health and Human Services guidelines published in Biosafety in
Microbiological and Biomedical Laboratories (stock no. 017-040-00547-4)
• Occupational Safety and Health Standards, Toxic and Hazardous Substances
(29 CFR
§
1910.1030).
Additional information about biohazard guidelines is available at:
http://www.cdc.gov
Follow all applicable local, state/provincial, and/or national regulations. Wear appropriate protective eyewear, clothing, and gloves.
Laser Safety
Laser Classification
The 3730/3730xl DNA Analyzer uses a laser. Under normal operating conditions, the instrument laser is categorized as a Class I laser. When safety interlocks are disabled during certain servicing procedures, the laser can cause permanent eye damage, and, therefore, is classified under those conditions as a Class IIIb laser.
The 3730/3730xl DNA Analyzer laser has been tested to and complies with the
“Radiation Control for Health and Safety Act of 1968 Performance Standard CFR 1040.”
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xix
Safety
Bar Code Scanner Laser Safety
The 3730/3730xl DNA Analyzer laser has been tested to and complies with standard
EN60825-1, “Radiation Safety of Laser Products, Equipment Classification,
Requirements, and User’s Guide.”
Laser Safety Requirements
To ensure safe laser operation:
• The system must be installed and maintained by an Applied Biosystems Technical
Representative.
• All instrument panels must be in place on the instrument while the instrument is operating. When all panels are installed, there is no detectable radiation present. If any panel is removed when the laser is operating (during service with safety interlocks disabled), you may be exposed to laser emissions in excess of the Class I rating.
• Do not remove safety labels or disable safety interlocks.
Additional Laser Safety Information
Refer to the user documentation provided with the laser for additional information on government and industry safety regulations.
LASER HAZARD. Lasers can burn the retina causing permanent blind spots. Never look directly into the laser beam. Remove jewelry and other items that can reflect the beam into your eyes. Do not remove the instrument top or front panels. Wear proper eye protection and post a laser warning sign at the entrance to the laboratory if the top or front panels are removed for service.
LASER BURN HAZARD. An overheated laser can cause severe burns if it comes in contact with the skin. DO NOT operate the laser when it cannot be cooled by its cooling fan. Always wear appropriate laser safety goggles.
Bar Code Scanner Laser Safety
Laser Classification
The bar code scanner included with the 3730/3730xl DNA Analyzer is categorized as a
Class II laser.
Laser Safety Requirements
Class II lasers are low-power, visible-light lasers that can damage the eyes. Never look directly into the laser beam. The scanner is designed to prevent human access to harmful levels of laser light during normal operation, user maintenance, or during prescribed service operations.
LASER HAZARD. Class II lasers can cause damage to eyes. Avoid looking into a Class
II laser beam or pointing a Class II laser beam into another person’s eyes.
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Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Safety
U.S. and Canadian Safety Standards
Computer Workstation Safety
Correct ergonomic configuration of your workstation can reduce or prevent effects such as fatigue, pain, and strain. Minimize or eliminate these effects by configuring your workstation to promote neutral or relaxed working positions.
MUSCULOSKELETAL AND REPETITIVE MOTION HAZARD. These hazards are caused by potential risk factors that include but are not limited to repetitive motion, awkward posture, forceful exertion, holding static unhealthy positions, contact pressure, and other workstation environmental factors.
• Use equipment that comfortably supports you in neutral working positions and allows adequate accessibility to the keyboard, monitor, and mouse.
• Position the keyboard, mouse, and monitor to promote relaxed body and head postures.
Safety and Electromagnetic Compatibility (EMC) Standards
U.S. and Canadian Safety Standards
This instrument has been tested to and complies with standard UL 3101-1, “Safety
Requirements for Electrical Equipment for Laboratory Use, Part 1: General Requirements.”
This instrument has been tested to and complies with standard CSA 1010.1, “Safety
Requirements for Electrical Equipment for Measurement, Control, and Laboratory Use,
Part 1: General Requirements.”
Canadian EMC Standard
This instrument has been tested to and complies with ICES-001, Issue 3: Industrial,
Scientific, and Medical Radio Frequency Generators.
European Safety and EMC Standards
Safety
This instrument meets European requirements for safety (Low Voltage Directive
73/23/EEC). This instrument has been tested to and complies with standards EN 61010-
1:2001, “Safety Requirements for Electrical Equipment for Measurement, Control and
Laboratory Use, Part 1: General Requirements” and EN 61010-2-010, “Particular
Requirements for Laboratory Equipment for the Heating of Materials.”
EMC
This instrument meets European requirements for emission and immunity (EMC
Directive 89/336/EEC). This instrument has been tested to and complies with standard
EN 61326 (Group 1, Class B), “Electrical Equipment for Measurement, Control and
Laboratory Use – EMC Requirements.”
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Safety
Safety and Electromagnetic Compatibility (EMC) Standards
Australian EMC Standards
This instrument has been tested to and complies with standard AS/NZS 2064, “Limits and Methods Measurement of Electromagnetic Disturbance Characteristics of Industrial,
Scientific, and Medical (ISM) Radio-frequency Equipment.”
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Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Preparing the Instrument
Workflow
Preparing the
Instrument
Performing
Spatial Calibration
Performing
Spectral Calibration for Sequencing and
Fragment Analysis
Setting Up the Software for
DNA Sequencing
Setting Up the Software for
Fragment Analysis
Running the Instrument
Performing
Maintenance
Audit Trails and Access Control
Notes
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Start the
3730/3730xl Data Collection
Software
Install the capillary array
Replace the polymer
Prepare buffer and fill the reservoirs
1
1
Chapter 1 Preparing the Instrument
Instrument and Parts
Instrument and Parts
Polymer Delivery Pump (PDP)
Behind the oven door
Capillary array
installing
Pump Block
maintenance
Polymer supply tube
Polymer reservoir
filling
installing
Anode buffer jar
filling
installing
Interconnect tube
Lower polymer block
cleaning
Interior light button
Status lights
description
description
Tray button Buffer reservoir
filling
Water reservoir
filling
Stacker door
Oven door
Waste reservoir
installing
Stacker door indicator light
Notes
2
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Polymer Delivery Pump Detail
Polymer Delivery Pump Detail
PDP motor
Mounting pin
Syringe fitting
Water seal
Waste fitting
Water trap
Piston
Mounting pin
Pump chamber
Pump block
Check valve
Polymer supply tube
Polymer supply bottle cap with hole
Mounting pin
O-ring
Electrode
PDP motor cover
Capillary array tip
Capillary array knob
Double-tapered ferrule
Array port
Interconnect tube
Buffer valve pin
Lower polymer block
Mounting pin
Overflow hole
Buffer fill-line
Capillary array
1
Buffer jar (67mL anode reservoir)
Polymer supply bottle
Notes
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
3
Chapter 1 Preparing the Instrument
Overview
Overview
This chapter explains how to prepare the instrument for a run by installing the capillary array, buffer, and reservoirs.
Powering On the Computer and
3730/3730xl Instrument
1.
Press the power button on the monitor to turn it on.
2.
Press the power button on the computer to turn it on.
3.
In the Log On to Windows dialog box:
a.
c.
In the User Name field, enter your user name.
b.
In the Password field, enter your password.
Click .
4.
Close the oven door.
5.
Close the stacker drawer.
3a
3b
Stacker drawer
6.
Close the instrument door.
Instrument door
7.
Wait until the monitor displays the desktop of the
Windows
®
operating system.
Notes
4
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Overview
Powering On the Computer and 3730/3730xl Instrument
8.
Press the power button on the 3730/3730xl instrument to turn it on.
Status
Status
Light
Action
• The instrument is ready
• An automated wizard operation is in progress with the instrument door closed
• A run is in progress solid green flashing green
Go to
• The instrument cannot communicate with the computer.
solid yellow
• The instrument is downloading firmware
• The instrument is performing diagnostics
• The oven door is open
• The instrument door is open
• The buffer reservoir is not installed
• The capillary array is not installed
• An automated wizard operation is in progress with the instrument door open flashing yellow
• The instrument has detected a problem solid red
Go to
Go to
Go to
00:00:10
Initializing
00:00:30
Downloading firmware
00:00:30
Performing diagnostics
Ready
Yes
Yes
No
No
Yes
>00:05:00
1
Notes
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
5
Chapter 1 Preparing the Instrument
Overview
Troubleshooting
The instrument displays a flashing yellow light.
Determine the source of the problem as follows:
1. Press on the instrument door to ensure that it is closed.
If the 3730/3730xl instrument displays the green status light, then the instrument door was open. Go
2. If the 3730/3730xl instrument continues to display the flashing yellow light: a. Open the instrument door.
b. Press on the oven door to verify that it is closed.
c. Close the instrument door.
If the 3730/3730xl instrument displays the green status light, then the oven door was open. Go to
3. If the 3730/3730xl instrument continues to display the flashing yellow light: a. Open the instrument door.
b. Open the oven door.
c. Check that the buffer reservoir and capillary array are installed. d. Close the oven door. e. Close the instrument door.
Instrument door
Oven door
OK – Go to
Capillary array (installed)
Buffer reservoir (installed)
OK – Go to
Capillary array (not installed)
Buffer reservoir (not installed)
Capillary array (installed)
Buffer reservoir (not installed)
Notes
6
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Overview
Powering On the Computer and 3730/3730xl Instrument
Troubleshooting (continued)
The Instrument displays a solid yellow light.
Determine the source of the problem as follows:
Verify that the ...
1. Monitor displays the desktop of the Windows operating system.
2. Ethernet cable is connected to the back of the 3730/3730xl instrument.
3. Other end of the Ethernet cable is connected to the computer.
4. Instrument door is closed.
5. Buffer, water, and waste reservoirs are in place.
6. 3730User account password is functional.
If the instrument continues to display the solid yellow light, contact Applied Biosystems technical support or your service representative for further assistance.
The Instrument displays a solid red light.
Determine the source of the problem as follows:
1. If the instrument continues to display the solid red light: a. Turn off the instrument.
b. Wait for 30 seconds.
c. Turn on the instrument.
2. If the instrument continues to display the solid red light: a. Start the 3730/3730xl Data Collection Software as
b. In the Tree pane of the Data Collection Software, double-click GA Instruments > ga3730 >
instrument name > Instrument Status >
Event Log.
c. In the Event Log view, find the last message in the log file.
d. Using the error code, perform the required tasks to fix the problem.
3. If the instrument continues to display the solid red light, contact Applied Biosystems technical support or your service representative for further assistance.
1
Notes
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
7
Chapter 1 Preparing the Instrument
Starting the 3730/3730xl Data Collection Software
Starting the 3730/3730xl Data Collection Software
1.
Select > Programs >
Applied Biosystems >
Unified Data Collection >
Run Unified Data Collection v2.0.
The data collection software opens the Service
Console dialog box.
00:00:00
Red circles indicate that applications of the data collection software are not running.
Wait for the Service Console dialog box to open the applications of the data collection software.
Not running Starting Running
When all applications are running (green squares), the Data Collection software opens the
Data Collection Viewer.
<00:01:00
>00:01:00
Applications of the data collection software are running
Notes
8
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Installing the Capillary Array
Installing a New or Used Capillary Array
Installing the Capillary Array
Required Materials
• Capillary array, 96- or 48-capillary
• Lab wipes, lint-free
• Gloves
CHEMICAL HAZARD. POP-7
polymer may cause eye, skin, and respiratory tract irritation. Please read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. Use for research and development purposes only.
Guidelines for Capillary Use
• Do not bend the capillaries
• Store capillary arrays using a buffer reservoir and the header shipping cover (for storing information see,
Installing a New or Used Capillary Array
IMPORTANT!
Wear gloves when you handle the capillary array.
Header shipping cover
Buffer reservoir
1.
Close the instrument door.
2.
In the Data Collection software, select
GA Instruments > ga3730 >
instrument name >.
Instrument door
1
3.
On the toolbar, select
Wizards > Install Array Wizard.
4.
Install the array as instructed by the Array
Wizard.
After Replacing the Capillary Array
• Perform a spatial calibration o
Notes
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
9
Chapter 1 Preparing the Instrument
Replacing the Polymer
Replacing the Polymer
Note:
This section may be skipped if you have installed a capillary array using the Install Array wizard during the initial activation of the instrument.
Required Materials
• POP-7 polymer
• Wipes, lint-free
• Gloves
Guidelines for Polymer Use
• Check the polymer blocks and lines daily for bubbles.
• Ensure that you have enough polymer for operation:
– A 96-capillary run uses approximately 250 µL of polymer
– A 48-capillary run uses approximately 110 µL of polymer.
CHEMICAL HAZARD. POP-7
polymer may cause eye, skin, and respiratory tract irritation. Please read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. Use for research and development purposes only.
When to Replace the Polymer
Replace the polymer on the instrument:
• Weekly (polymer lifetime is 7 days at 25 °C)
• If insufficient polymer remains for the planned run set
IMPORTANT!
Failure to replace expired/old polymer may lead to loss of resolution and data quality.
POP-7 Storage
Su M T W Th F S
IMPORTANT!
Wear gloves when you handle polymer.
2
°C to 8 °C
Until expiration date on the bottle
Notes
10
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
1.
Close the instrument door.
2.
In the Data Collection software, select
GA Instruments > ga3730 >
instrument name >.
3.
On the toolbar, select Wizards > Change
Polymer Wizard.
4.
Change the polymer as instructed by the Change
Polymer wizard.
Replacing the Polymer
Installing a New or Used Capillary Array
Instrument door
1
Notes
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
11
Chapter 1 Preparing the Instrument
Preparing Buffer and Filling the Reservoirs
Preparing Buffer and Filling the Reservoirs
Required Materials
• Retainer, buffer/water/waste
• Septa
• Reservoir caps
• Reservoir, buffer/water/waste
• Plate base, water/waste
• Plate base, buffer
• Water, deionized, 180 mL plus, 160 mL for water and waste reservoirs
• 10X Genetic Analyzer Running Buffer with
EDTA, 20 mL
• Graduated cylinder, 250-mL
• Gloves, silicone-free, powder-free
Guidelines for Buffer Use
The 1X run buffer can be stored at:
• 2 to 8 °C for up to 1 month
• Room temperature for 1 week
CHEMICAL HAZARD. Running
Buffer with EDTA. Please read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.
Buffer Storage Conditions
Option A Option B
2
°C to
8
°C
20
°C to
25
°C
When to Change the Buffer, Water, and Waste
Replace the buffer in the reservoirs every 48 hours, or before each batch of runs.
Su M T W Th F S Su M T W Th F S
1X
3730
Run
Buffer
IMPORTANT!
Failure to replace buffer may lead to loss of resolution and data quality.
1 month 7 days
Notes
12
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Preparing Buffer and Filling the Reservoirs
Preparing the 1X Run Buffer
Preparing the 1X Run Buffer
IMPORTANT!
Wear gloves when you handle running buffer with EDTA.
1.
Add 20 mL 10X running buffer with EDTA into a graduated cylinder.
2.
Add 180 mL (qs) deionized water to bring the total volume to 200 mL.
3.
Mix well and set aside.
Filling the Water and Buffer Reservoirs
200 mL total
180 mL DI water
20 mL 10X Running
Buffer with EDTA
IMPORTANT!
Wear gloves when you handle the reservoir.
1.
Close the instrument door.
2.
Press the Tray button to bring the autosampler to the forward position.
3.
Wait for the autosampler to stop moving and for the green status light to illuminate, before you open the instrument door.
4.
Unplug the buffer reservoir. Remove the buffer, water, and waste reservoir assemblies from the instrument.
5.
Disassemble each reservoir assembly and empty the contents of the reservoirs into an aqueous waste container.
Status lights
Instrument door
Tray button
Aqueous
Waste
1
Notes
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
13
Chapter 1 Preparing the Instrument
Preparing Buffer and Filling the Reservoirs
6.
Rinse each reservoir using deionized water.
7.
Dry the reservoirs using lint-free wipes.
DI H
2
O
≤ 40 °C
Notes
14
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Preparing Buffer and Filling the Reservoirs
Filling the Water and Buffer Reservoirs
8.
Fill and assemble the reservoirs.
Buffer Reservoir Assembly
1. Add 80 mL 1X run buffer to the Buffer reservoir.
2. Assemble the reservoir assembly as shown below:
Retainer
Septa
Reservoir cap
Reservoir
Heated plate base
Power cable
GR2210
9.
To prevent damage to the capillary array, inspect each reservoir assembly and verify that the:
• Septa fits snugly and flush on the reservoir cap
• Rubber gasket around the edge of the reservoir cap is seated correctly
Water and Waste Reservoir Assemblies
1. Add 80 mL high-quality deionized water to each reservoir (Water and Waste).
2. Assemble each reservoir assembly as shown below:
Retainer
Septa
Reservoir cap
Reservoir
Plate base
• Holes of the plate retainer and the septa strip are aligned
Rubber gasket not seated correctly
Plate retainer holes and septa holes are not aligned
1
10.
Dry the reservoirs using lint-free wipes.
Lint-free wipe
Notes
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
15
Chapter 1 Preparing the Instrument
Placing Reservoirs into the Instrument
Placing Reservoirs into the Instrument
1.
Connect the Buffer reservoir plate base cable into the heater outlet within the instrument.
Heater outlet
Plate base cable
2.
Move the buffer reservoir to the Buffer position
(left).
IMPORTANT!
After placing the buffer reservoir, make sure the cable is out of the way of the autosampler.
3.
Place the Water and Waste reservoirs into the instrument. The reservoirs must be in the following order from left to right:
a.
b.
Buffer reservoir
Water reservoir
c.
Waste reservoir
4.
Close the instrument door.
3a 3b 3c
Buffer reservoir
Buffer position
Instrument door
5.
Press the tray button to return the autosampler to the array position.
Tray button
Notes
16
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Placing Reservoirs into the Instrument
Filling the Anode Buffer Jar
Filling the Anode Buffer Jar
Change the anode buffer:
• Before each group of scheduled runs, or at least every 24 to 48 hours
• Every time you fill the polymer block with new polymer
• Every time you change the buffer reservoir
IMPORTANT!
Wear gloves when you handle the anode buffer jar.
1.
Remove the anode buffer jar by pulling down while twisting slowly.
2.
Empty the anode buffer jar into an aqueous waste container.
3.
Rinse the anode buffer jar using deionized water.
4.
Rinse the anode buffer jar using 1X run buffer:
a.
Add 5 mL 1X run buffer to the anode buffer jar.
b.
Tilt the anode buffer jar 90
°.
c.
Rotate the jar to rinse the interior with buffer.
d.
Empty the anode buffer jar into an aqueous waste container.
5.
Add 67 mL 1X run buffer to the jar.
90
°
Aqueous
Waste
4b
4c
4d
Aqueous
Waste
Notes
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
17
1
Chapter 1 Preparing the Instrument
Placing Reservoirs into the Instrument
6.
Put the anode buffer jar on the instrument with the overflow hole facing you.
Note:
The meniscus should line up just under the red fill line when installed on the instrument.
7.
Verify that the electrode is immersed in the buffer.
8.
If the reservoir fills completely as polymer is
added, perform steps 1 through
procedure to discard and replace the running buffer.
Note:
Replace buffer if excess polymer is expelled into the anode jar.
Meniscus
Electrode
Notes
18
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Performing Spatial Calibration
Workflow
Preparing the
Instrument
Performing
Spatial Calibration
Performing
Spectral Calibration for Sequencing and
Fragment Analysis
Setting Up the Software for
DNA Sequencing
Setting Up the Software for
Fragment Analysis
Running the Instrument
Performing
Maintenance
Audit Trails and Access Control
Notes
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Perform spatial calibration
Evaluate the calibration data
19
2
Chapter 2 Performing Spatial Calibration
Overview
Overview
What a Spatial Calibration Tells You
The 3730/3730xl Data Collection Software uses images collected during spatial calibration to establish a relationship between the signal emitted by each capillary and the position where that signal falls and is detected by the CCD camera.
When to Perform the Spatial Calibration
Perform a spatial calibration after you:
• Install a new or used capillary array
• Remove the capillary array from the detection cell block (even to adjust it)
• Move the instrument (even if the instrument was moved on a table with wheels)
Performing Spatial Calibration
1.
In the Tree pane of the Data Collection Software, double-click GA Instruments > ga3730 >
instrument name > Spatial Run Scheduler.
Spatial Run Scheduler
2.
In the Spatial Run Scheduler view, do one of the following:
• If the capillaries contain fresh polymer, select Protocol > SpatialNoFill.
• Otherwise, select Protocol > SpatialFill.
Note:
You do not need to fill the capillaries each time you perform a spatial calibration.
Notes
20
Select
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
3.
Click .
The approximate calibration run times are:
• 48-cap/36cm array with fill, 4 minutes.
• 96-cap/36cm array with fill, 3 minutes.
• No fill, 2 minutes.
4.
Evaluate the calibration as explained on
.
Performing Spatial Calibration
When to Perform the Spatial Calibration
Spatial Run Scheduler
2
Notes
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
21
Chapter 2 Performing Spatial Calibration
Evaluating the Calibration Data
Evaluating the Calibration Data
Note:
Examples of passing spatial calibration profiles start on
.
1.
Verify that the peaks of the spatial are approximately the same height.
Are the peaks in the profile approximately the same height?
Yes – Go to
No – How does the peak height vary?
• If the peak height increases at the beginning and the end of the spatial profile, then the variation in peak height is acceptable.
Go to
Irregular – If the peak heights are irregular, go to
“If the Calibration Fails” on page 25
.
Magnifying the Spatial Profile
a. Click and drag the cursor to create a box around the area of interest.
b. Release the mouse button.
The data collection software displays the selected region.
c. Press R to reset the view.
2.
Verify that an orange cross appears at the top of each peak in the profile.
Does a cross appear at the top of each peak?
No – Where in the profile is the peak located?
Notes
22
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
• Left side of the profile
If using a 96-capillary array, a small peak may appear in the left side of the profile.
The peak is normal, go to step 3
.
• After the first peak
The data collection software did not locate the peak correctly.
Move an orange cross to cover the peak.
See,
“To move an orange cross:” on page 24 .
Evaluating the Calibration Data
When to Perform the Spatial Calibration
Peak does not contain an orange cross
2
3.
Check the profile for irregular peaks.
Does the profile contain any irregular peaks?
Yes – The calibration run has failed. Go to
Calibration Fails” on page 25 .
.
Elements of a poor spatial
4.
Examine each row of the 96 Capillary Position table. Typical values for the Left spacing and
Right spacing columns are:
• 4 to 8 pixels for a 96-capillary array
• 9 to 11 pixels for a 48-capillary array
Note:
Values greater than those stated above are acceptable if you are able to see a corresponding gap in the capillaries in the detection cell.
Be sure to account for all capillaries (e.g., 96 capillary positions for 96 capillary array).
– If not, verify that all peaks have crosses. If each peak does not each have a cross, see the
Troubleshooting table below.
– If yes, go to step 5.
Left spacing and Right spacing columns
Notes
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
23
Chapter 2 Performing Spatial Calibration
Evaluating the Calibration Data
5.
Accept or reject the spatial calibration as follows:
If the calibration:
• Passed, click writes the calibration data to the database.
• Failed, click , then go to
Calibration Fails” on page 25 .
Accept and
Reject buttons
Troubleshooting
Peak does not contain an orange cross.
Note:
The cross positions cannot be altered after you have accepted the calibration data.
To move an orange cross:
1. Magnify the view of the peak without a cross.
2. Determine the peak pixel position.
3. Change the value for the incorrectly positioned cross.
4. Click outside of that box.
Change the cross position
Notes
24
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Evaluating the Calibration Data
When to Perform the Spatial Calibration
Troubleshooting
If the Calibration Fails
If the calibration failed, or if you do not like the appearance of the profile, try one or more of the following actions:
CHEMICAL HAZARD. Methanol is a flammable liquid and vapor. Exposure causes eye and skin irritation, and may cause central nervous system depression and nerve damage. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.
and repeat the spatial calibration.
2. If the calibration fails again: a. Follow the Bubble Remove wizard to remove bubbles and to fill the capillaries with polymer.
to repeat the spatial calibration.
3. If the calibration fails again: a. Open the Instrument door.
b. Open the oven door.
c. Open the detection cell door and turn the cam knob 1/4 turn clockwise (pointer left).
GR2193
Front surface of the detection cell d. Pull the pump and lower polymer blocks forward until the detection cell comes out of the detection block.
e. Remove from the pump block:
– Tip of the capillary array
– Array knob
– Ferrule f. Add one drop of methanol to a sterile swab or lintfree wipe and apply to the front surface of the detection cell.
g. Gently clean the front surface of the detection cell using the sterile swab or lint-free wipe.
h. Replace the tip of the capillary array, array knob, and ferrule into the pump block.
i. Push the pump and lower polymer blocks back against the pump panel, making sure that the buffer valve lever properly engages the buffer pin valve.
j. Carefully place the detection cell into the detection block and secure it by rotating the cam knob 1/4 turn counterclockwise (pointer down).
k. Close the detection cell door.
l. Close the oven door.
m. Close the Instrument door.
n. Using the Bubble Remove wizard, remove all bubbles. Pay particular attention to the array port area.
to repeat the calibration.
2
Notes
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
25
Chapter 2 Performing Spatial Calibration
Evaluating the Calibration Data
Troubleshooting
4. If the calibration fails again: a. Perform steps 3a through 3c.
b. Reposition the capillary array window in the detection cell.
c. Perform steps 3j through 3m.
to repeat the calibration.
5. If calibration fails again, replace the capillary array as explained in
“Installing the Capillary Array” on page 9
.
Notes
26
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Evaluating the Calibration Data
Examples of Passing Spatial Profiles
Examples of Passing Spatial Profiles
IMPORTANT!
Improper peak identification may lead to sample mistracking on the instrument, and potential sample misnaming.
Passing Profile #1
This example shows a typical passing profile.
2
Passing Profile #2
Passing Profile #3
Background artifact
This example shows a passing profile with high artifactual background at the left margin.
Notes
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
27
Chapter 2 Performing Spatial Calibration
Evaluating the Calibration Data
Notes
28
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Performing Spectral Calibration For
Sequencing and Fragment Analysis
Workflow
Preparing the
Instrument
Performing
Spatial Calibration
Performing
Spectral Calibration for Sequencing and
Fragment Analysis
Setting Up the Software for
DNA Sequencing
Setting Up the Software for
Fragment Analysis
Running the Instrument
Prepare the spectral calibration chemistry
Create a spectral instrument protocol
Perform spectral calibration
Performing
Maintenance
Evaluate the spectral calibration data
Audit Trails and Access Control
Notes
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
29
3
Chapter 3 Performing Spectral Calibration For Sequencing and Fragment Analysis
Overview
Overview
A spectral calibration creates a matrix that is used during a run to reduce raw data from the instrument to the 4- or 5-dye data stored in the sample files. Performing a spectral calibration is similar to performing a sample run, except that calibration standards are run in place of samples, and a spectral calibration module is used in place of a run module.
IMPORTANT!
Do not run your computer operating systems’ Internet Connection Wizard during a spectral calibration.
Note:
A spectral calibration algorithm checks dye order. The error message if the algorithm determines that the dyes are in the incorrect order is, "failed calibration due to bad data: Bad dye order detected."
Spectral calibrations are performed with a specific combination of:
• Dye set (G5, G5-RCT, Any4Dye, Any5Dye, E or Z). For further information see,
“Preparing the Spectral Calibration Chemistry” on page 32 and,
G5-RCT, Any4Dye, and Any5Dye Dye Sets .
• Array type (48-capillary or 96-capillary)
• Array length (36-cm or 50-cm)
IMPORTANT!
Spectrals must be calibrated for dye set, array type, and array length.
When to Perform the Calibration
Perform a spectral calibration:
• Whenever you use a new dye set on the instrument
• After the laser or CCD camera has been realigned/replaced by a service engineer
• If you see a decrease in spectral separation (pull-up and/or pull-down peaks)
• If you alter any condition (dye set, array type, or array length)
Changing
Capillary Array
Lengths
For each dye set, a single spectral calibration can not be used for all capillary array lengths .
• For every sequencing dye set, you must create a separate spectral calibration for each capillary array length and array type.
• For every fragment analysis dye set, you must create a separate spectral calibration for each capillary array length and array type.
Refer to
for information on how to switch calibrations.
Notes
30
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Overview
Required Materials
Required
Materials
Part numbers are located in Appendix A
.
Description
ABI P
RISM
®
BigDye
®
Terminator v3.1 or v1.1 Sequencing Standard or, DS-33
Matrix Standard
ABI P
RISM
®
384- or 96-Well Reaction Plate w/ Barcode
Multichannel pipettor
Plate retainer
• Plate septum with black plate base or,
• Heat-seal with gray plate base
Hi-Di
™
formamide
Heated block or thermal cycler
Container with ice
Centrifuge with microplate adapter
Microcentrifuge
Vortex
Gloves
Two Types of
Calibration
Standards
Two types of calibration standards are used to create a matrix:
For Fragment Analysis:
• Matrix standards – four or five fragments of varying size are individually labeled with one of the four or five dyes of a set.
For Sequencing:
• Sequencing Standards – standard sequencing reaction fragments of varying size are individually labeled with one of the four dyes.
Use the tables below to determine the correct dye set and calibration standard for the application you are using.
Sequencing Chemistry
ABI P
RISM
®
BigDye
®
v3.1Terminator
ABI P
RISM
®
BigDye ® v1.1 Terminator
.
Fragment Analysis Chemistry
ABI P
RISM®
Linkage Mapping Set v2.5/custom oligos
ABI P
RISM®
Linkage Mapping Set v2.5/custom oligos
Dye Set Calibration Standards
Z_BigDyeV3
BigDye ® v3.1 Terminator Sequencing Standard
E_BigDyeV1
BigDye ® v1.1 Terminator Sequencing Standard
Dye Set
G5
G5-RCT
Calibration Standards
DS-33
DS-33
3
Notes
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
31
Chapter 3 Performing Spectral Calibration For Sequencing and Fragment Analysis
Preparing the Spectral Calibration Chemistry
Preparing the Spectral Calibration Chemistry
CHEMICAL HAZARD.
Formamide causes eye, skin, and respiratory tract irritation. It is a possible reproductive and birth defect hazard. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.
1.
Dilute spectral calibration standard with Hi-Di
™ formamide according to the insert instructions.
BigDye
®
Terminator v3.1 or v1.1 Sequencing Standard or, for fragment analysis,
DS-33 matrix standard
Dilute with Hi-Di formamide
2.
Vortex thoroughly.
Vortex
00:00:05
3.
Briefly centrifuge the mixture.
<1500
×g
00:00:05
4.
Heat the standard tube at 95 °C for 5 minutes to denature the DNA.
95
°C
00:02:00
− 4 °C
5.
Cool the tubes on ice for 2 minutes.
6.
Vortex thoroughly and then briefly centrifuge the mixture.
Notes
00:02:00
32
Denatured standard
Prepared standard
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Sealing and Preparing the Plate
Assemblies
WARNING Do not use warped or damaged plates.
1.
Add the denatured standard to the wells of a 384- or 96-well reaction plate:
If using a:
• 48-capillary, 96-well plate – Add 10 µL of denatured standard to each well.
• 384-well plate – Add 5 µL of denatured standard into alternating wells of the plate.
See
“Default Load Maps” on page 125
2.
Seal the plate with septum or heat-seal:
With septum:
a.
Place the plate on a clean, level surface.
b.
Lay the septum flat on the plate.
c.
Align the holes in the septum strip with the wells of the plate, then firmly press downward onto the plate.
Preparing the Spectral Calibration Chemistry
Sealing and Preparing the Plate Assemblies
96-Well plate
384-Well plate
Add 10 µL prepared standard to each well
Plate septum
(384- or 96-well)
Sample plate
(384- or 96-well)
Add 5 µL prepared standard into alternating wells (wells A1,
C1, E1, …)
3
Notes
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33
3.
Chapter 3 Performing Spectral Calibration For Sequencing and Fragment Analysis
Preparing the Spectral Calibration Chemistry
d.
Ensure that:
• The septa lie flat against the plate. You should not feel any lumps or raised edges.
• The septa are inserted straight into the wells. You should not see any bent or crooked duckbills when viewing the plate from above.
With heat-seal:
a.
Follow your thermal sealer instrument instructions.
Briefly centrifuge the plate.
Septum and well not aligned
Septum and well not aligned
Septum and well not aligned
Septum and well aligned
Prepared standard
<1500
× g
0:05
4.
Remove the plate from the centrifuge and verify that each sample is positioned correctly in the bottom of its well.
If the reagents of any well contain bubbles or are not located at the bottom of the well, repeat steps
Sample is at the bottom of the well
Notes
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Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Preparing the Spectral Calibration Chemistry
Sealing and Preparing the Plate Assemblies
5.
Assemble the plate assembly as shown below.
Septum Assembly
Plate retainer
Plate septum
Septum-sealed sample plate
Black
plate base
Plate retainer
Heat-sealed Assembly
Heatsealed sample plate
Gray plate base
Assembled components
Assembled components
Use only black plate bases with septa-sealed plates.
Use only gray plate bases with heat-sealed plates.
6.
Verify that the holes of the plate retainer and the septa are aligned.
IMPORTANT!
The plate may damage the array if the retainer and the septum holes are not aligned.
IMPORTANT! Heat Seal Recommendations
• Use 3-mil Applied Biosystems heat seal film (PN 4337570). This film is 3-mil before, and 1-mil after, heating.
• Do not use heat seal film thicker than 1-mil, after heating, on 3730/3730xl DNA Analyzer.
• Do not use heat-seal film containing adhesives or metals as these may damage the instrument’s piercing needles.
3
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35
Chapter 3 Performing Spectral Calibration For Sequencing and Fragment Analysis
Creating a Spectral Instrument Protocol
Creating a Spectral Instrument Protocol
1.
In the Tree pane of the Data Collection Software, click GA Instruments > ga3730 >
Protocol Manager.
Create instrument protocols here
Create analysis protocols here
2.
In the Instruments Protocols pane, click
The Protocol Editor dialog box opens.
.
3.
Select Spectral from the Run Module drop-list.
Notes
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Applied Biosystems 3730/3730xl DNA Analyzer User Guide
4.
The Protocol Editor now displays additional drop-lists.Select from the following:
If you are using a matrix standard for spectral calibration, choose:
a.
b.
Run Module: Spect36_MtxStd_1
Array Length: 36
c.
Chemistry: matrixStandard
IMPORTANT!
Select the appropriate capillary array length. The array length must match the array length information from the Install Array wizard.
If you are using a sequencing standard for spectral calibration you may choose 36-cm or
50-cm array length:
36-cm capillary array:
a.
Run module: Spect36_SeqStd_1
b.
Chemistry: sequenceStandard
50-cm capillary array:
a.
Run module: Spect50_SeqStd
b.
Chemistry: sequenceStandard
Note:
The Chemistry file for fragment analysis dye sets automatically defaults to the Matrix
Standard.
IMPORTANT!
Select the appropriate capillary array lengths. The array length must match the array length information from the Install Array wizard.
Dye Set
Z_BigDyeV3
E_BigDyeV1
Standard Type
BigDye
®
v3.1 Terminator Sequencing Standard
BigDye ® v1.1 Terminator Sequencing Standard
Creating a Spectral Instrument Protocol
Sealing and Preparing the Plate Assemblies
Chemistry File
Sequence Standard
Sequence Standard
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3
Chapter 3 Performing Spectral Calibration For Sequencing and Fragment Analysis
Creating a Spectral Instrument Protocol
Dye Set
G5
G5-RCT
Matrix Standard Set
DS-33
DS-33
Chemistry File
Matrix Standard
Matrix Standard
IMPORTANT!
Failure to select the correct chemistry file for the spectral calibration samples you are using results in a failing spectral run.
5.
(Optional) Click Edit Param to display the
Spectral Params dialog box.
Use this dialog box to edit the selection criteria for passing or failing spectral calibrations.
Parameters Valid Data Ranges*
Matrix Condition Number Bounds
Locate Start Point
Limit Analysis (scans)
Sensitivity
Minimum Quality Score
Lower: 1-10 Upper: 3-20
After Scan: 100-5000 Before Scan: 100-5000
400-20,000
0-0.9
.80-.99
*These ranges are dye-set independent
IMPORTANT!
Default parameter values are optimized and are recommended for most situations
Notes
38
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Creating a Spectral Calibration Plate Record
Creating a Spectral Calibration Plate Record
1.
In the Tree pane of the Data Collection Software, double-click
GA Instruments > ga3730 >
instrument name > Plate Manager.
2.
Click New to create a new plate.
3
3.
Complete the New Plate dialog box:
a.
b.
Enter ID or Barcode number
Enter a name for the plate.
c.
d.
e.
Optional: Enter a description for the plate record.
In the Application drop-list, select Spectral
Calibration.
In the Plate Type drop-list, select 96-Well or
384-Well.
f.
Enter desired scheduling. For more
information see, “Globally Modifying a
Notes
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
39
Chapter 3 Performing Spectral Calibration For Sequencing and Fragment Analysis
Creating a Spectral Calibration Plate Record
g.
In the Plate Sealing drop-list, select Septa or Heat Seal.
h.
Enter a name for the owner.
i.
j.
Enter a name for the operator.
Click .
4.
In the Spectral Calibration Plate Editor dialog box, enter the following information:
Note:
This example assumes that you are loading the first quadrant.
a.
b.
In the Sample Name column of row A01, enter a sample name, then click the next cell.
In the Comments column of row A01, enter any additional comments or notations for the sample at the corresponding position of the plate.
c.
In the Instrument Protocol 1 column of row A01, select a protocol from the droplist.
5.
Highlight the entire row.
6.
Select Edit > Fill Down Special.
Based on the plate type (96- or 384-well) and capillary array (48 or 96 capillaries) you are using, select the appropriate fill down option:
– 96 capillary/96-well plate: Fill Down
– 48 capillary/96-well plate: Fill down Special
(48 Cap)
– 96 capillary/384-well plate: Fill down Special
(96 Cap)
– 48 capillary/384-well plate: Fill down Special
(48 Cap)
7.
Click .
You have successfully created a plate record for the spectral calibration plate.
5a
5b 5c
Notes
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Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Loading the Plate into the Instrument
1.
The name of the plate you just created is displayed in the Input Stack window of the Data
Collection software, and is ready to run.
2.
Open the stacker drawer.
3.
Open the In Stack tower door.
Loading the Plate into the Instrument
Stacker drawer
4.
Place the plate assembly into the stacker.
IMPORTANT!
When placing the plate into the stacker, the plate must be oriented so that the notched corner of the plate assembly is located in the rear-right corner of the stacker.
5.
Close the In Stack tower door.
6.
Close the Stacker drawer.
Notched corner of the plate assembly
3
In Stacker tower door
Notes
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41
Chapter 3 Performing Spectral Calibration For Sequencing and Fragment Analysis
Running the Spectral Calibration Plate
Running the Spectral Calibration Plate
1.
In the Tree pane of the Data Collection Software, double-click
GA Instruments > ga3730 >
instrument name > Run Scheduler.
2.
In the Run Scheduler view:
a.
In the Add Plate field, scan the barcode of a plate to add it to the input stack.
.
b.
Or,
Type the plate ID and press Enter to add it to the input stack.
3.
In the toolbar of the Data Collection Software window, click to begin the run.
4.
The Processing Plates dialog box opens, then click .
Note:
The instrument may pause before running the plate to raise the oven temperature.
Application
Sequencing
Sequencing
Fragment Analysis
Capillary Array Length (cm) Approximate Spectral Run Time
50
36
36
120
60
32 a. The data collection software may take up to 30 min to calculate the matrices after the run.
a
(min)
5.
When the run is finished, remove the plate from the instrument.
A9009DR5
Notes
42
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Viewing the Pass/Fail Status After the Run
After the instrument completes the spectral calibration run, the pass or fail status of each capillary is recorded in the Events Messages section of the Instrument
Status window.
1.
In the Tree pane of the Data Collection Software, click GA Instruments > ga3730 >
instrument
name > Instrument Status > Event Log.
Running the Spectral Calibration Plate
Viewing the Pass/Fail Status After the Run
3
Notes
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43
Chapter 3 Performing Spectral Calibration For Sequencing and Fragment Analysis
Running the Spectral Calibration Plate
2.
In the Events Messages section of the window, view the status of each capillary.
Cap # Pass/fail status Q-value
Condition number
Dye set G5 status results
For a good-quality calibration, each capillary should have a:
• Q-value:
– above 0.95 for matrix standards
– above 0.93 for sequence standards
• Condition number within range of:
Dye Set
Default Condition
Number Range
Sequencing Analysis
Z_BigDyeV3 2.5 to 4.5
E_BigDyeV1
Fragment Analysis
3.0 to 5
G5
G5-RCT
9.5 to 14.5
9.5 to 14.5
Notes
44
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Evaluating the Spectral Calibration Data
Viewing the Pass/Fail Status After the Run
Evaluating the Spectral Calibration Data
IMPORTANT!
Review and evaluate the spectral calibration profile for each capillary, even if the Spectral
Calibration Results box indicated that they all passed.
Note:
50 contain examples of passing sequencing spectral calibration profiles, and
contains an example of a passing fragment analysis spectral calibration profile.
1.
In the Tree pane of the Data Collection Software, click GA Instruments > ga3730 >
instrument name > Spectral Viewer.
3
Plate diagram
Spectral profile
Raw data
(matrix standards)
Rename or set the active spectral calibration here
Notes
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
45
Chapter 3 Performing Spectral Calibration For Sequencing and Fragment Analysis
Evaluating the Spectral Calibration Data
2.
In the Dye Set drop-list, select the dye set you just created.
3.
Select a well on the plate diagram to view the spectral results of associated capillary.
Well A1
Selected well
Capillary status:
Passed (dark green)
Selected (light green)
Borrowed/Failed (tan)*
* Overridden capillaries are also tan, even if they originally passed.
4.
Evaluate the spectral calibration profile for the selected capillary:
a.
Verify that the order of the peaks in the spectral profile from left to right are:
– 4-dye: blue-green-yellow-red
– 5-dye: blue-green-yellow-red-orange
Do the peaks in the profile appear in the correct order?
Yes: Go to
No: The calibration run has failed. Go to
Green Yellow Red
Example of a 4-dye spectral calibration profile
Blue Green Yellow Red Orange
Example of a 5-dye spectral calibration profile
Notes
46
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Evaluating the Spectral Calibration Data
Viewing the Pass/Fail Status After the Run
b.
Verify that the peaks in the spectral profile do not contain gross overlaps, dips, or other irregularities (see
Spectral Profile” on page 48 ).
Are the peaks in the spectral profile separate and distinct?
Yes – The capillary has passed. Go to step 5.
No – The calibration run has failed. Go to
c.
Verify that the order of the peaks in the raw data profile from left to right are:
Fragment Analysis
– 5-dye: orange-red-yellow-green-blue
Are the peaks in the wrong order or are there any extraneous peaks that adversely affect the spectral profile?
Yes: The calibration run has failed. Go to
No: Go to
Dip
Example of a 4-dye sequencing raw data profile
Left to right: Orange, Red, Yellow, Green, Blue
3
5.
Repeat steps 3 and 4 for each capillary in the array.
Example of a 5-dye fragment analysis raw data profile
Peak is distinct and regular
Notes
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
47
Chapter 3 Performing Spectral Calibration For Sequencing and Fragment Analysis
Evaluating the Spectral Calibration Data
6.
Rename the spectral run. The spectral file default name is the day, date and time of the run.
a.
b.
c.
Click .
In the Rename Calibration dialog box, enter a descriptive name for the spectral calibration including the dye set, array length and polymer type (optional).
Click .
Tip: Magnifying the Spectral Profile
1. In the Tree pane of the Data Collection
Software, click
GA Instruments >
instrument name >
Viewer.
ga3730 >
Spectral
2. In the profile or raw data display, click - drag the cursor to create a box around the area of interest.
3. Release the mouse button.
The data collection software displays the selected region.
4. Press R to reset the view.
Selecting an area to magnify in a spectral profile
Magnified area of that spectral profile
Notes
48
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Examples of Passing Sequencing Spectral Calibrations
Examples of Passing Sequencing Spectral Calibrations
Dye Set Z Created from a Sequencing Standard
3
Notes
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
49
Chapter 3 Performing Spectral Calibration For Sequencing and Fragment Analysis
Examples of Passing Sequencing Spectral Calibrations
Dye Set E Created from a Sequencing
Standard
Notes
50
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Example of a Passing Fragment Analysis Spectral Calibration
Dye Set G5 Created from Matrix Standard Set DS-33
Example of a Passing Fragment Analysis Spectral
Calibration
Dye Set G5 Created from Matrix Standard
Set DS-33
3
Notes
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51
Chapter 3 Performing Spectral Calibration For Sequencing and Fragment Analysis
Spectral Viewer
Spectral Viewer
Selecting Active Spectral Calibrations
For best quality data, we suggest that you perform spectral calibrations every time a new array is installed in the instrument. However, you may choose to reuse previous spectral calibrations to apply to new data that will be generated on the instrument. Once data is collected, you cannot reapply a different spectral calibration.
IMPORTANT!
It is essential that you perform a spectral calibration any time the capillary array is moved or replaced when using DyeSetG5-RCT.
IMPORTANT!
If you installed an array that is a different length or type (48 vs 96) than you were using previously and if a previous spectral calibration for the new array/new conditions exits, you must reset the active spectral calibration. if a previous spectral for those array conditions exist. Otherwise, you must run a new spectral calibration.
Poor quality data or failed analyses are results of using the wrong spectral calibration.
IMPORTANT!
Spectrals must be calibrated for dye set, array type, and array length.
When a new spatial calibration is saved, the current spectral calibration for DyeSet G5-RCT is deactivated. Dye sets G5, E, and Z are not deactivated. If you wish to continue without a spectral recalibration, you may “Set” an active spectral using the instructions below.
All calibrations for your current dye set are listed in the List of Calibrations drop-list. Therefore, you can choose a spectral to use from that list prior to the beginning of a new run.
Note:
An asterisk * precedes failing calibrations.
Note:
The most recent spectral for each dye set is automatically chosen as the active calibration.
Each dye set can have its own active calibration. Thus, there is no need to manually set the active calibration if you are performing runs with various dye sets.
Notes
52
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
To select a previous spectral calibration:
1.
Select the dye set of interest.
2.
In the Spectral Viewer, click the List of
Calibrations drop-menu in the lower, right pane.
Spectral Viewer
Selecting Active Spectral Calibrations
Current calibration
Drop-list of previous calibrations for the current dye set
3
3.
Select the spectral calibration you want to use for future runs.
4.
Click Set to display your chosen spectral calibration in the Active Calibration text box.
Notes
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
53
Chapter 3 Performing Spectral Calibration For Sequencing and Fragment Analysis
Spectral Viewer
5.
(Optional) Click Rename to display the Rename
Calibration dialog box, enter a new name, and click OK.
Notes
54
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Troubleshooting
Selecting Active Spectral Calibrations
Troubleshooting
Troubleshooting spectral calibration
Observation
No signal.
If the spectral calibration fails, or if a message displays “No candidate spectral files found.”
Spikes in the data.
Possible Cause
Incorrect sample preparation.
Air bubbles in sample tray.
Clogged capillary.
Insufficient filling of array.
Expired spectral standards.
Expired polymer.
Air bubbles, especially in the polymer.
Possible contaminant in the polymer.
Recommended Action
Replace samples with fresh samples prepared with fresh Hi-Di™ formamide.
CHEMICAL HAZARD.
Formamide
causes eye, skin, and respiratory tract irritation. It is a possible reproductive and birth defect hazard. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.
Centrifuge samples to remove air bubbles.
Refill the capillaries using manual control. Look for clogged capillaries during capillary fill on the cathode side.
Check for broken capillaries and refill the capillary array.
Check the expiration date and storage conditions of the spectral standards. If necessary, replace with a fresh lot.
Replace the polymer with a fresh lot using the Change Polymer Wizard.
!
WARNING
CHEMICAL HAZARD.
POP-7 polymer
cause eye, skin, and respiratory tract irritation. Read the
MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.
• Refill the capillaries using the
Bubble Remove wizard.
• Properly bring the polymer to room temperature.
• Replace expired polymer.
Replace the polymer using the Change
Polymer wizard.
3
Notes
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55
Chapter 3 Performing Spectral Calibration For Sequencing and Fragment Analysis
Troubleshooting
Notes
56
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Setting Up the Software for DNA Sequencing
Workflow
Preparing the
Instrument
Performing
Spatial Calibration
Performing
Spectral Calibration for Sequencing and
Fragment Analysis
Setting Up the Software for
DNA Sequencing
Setting Up the Software for
Fragment Analysis
Running the Instrument
Performing
Maintenance
Audit Trails and Access Control
Notes
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Create required settings for automated sequencing analysis
Create and complete a sequencing analysis plate record
Fill down special
57
4
Chapter 4 Setting Up the Software for DNA Sequencing
Plate Records and Sequencing Analysis
Plate Records and Sequencing Analysis
Overview
A plate record is similar to a sample sheet or an injection list that you may have used with other Applied Biosystems instruments.
Important Notes
• A unique name must be assigned to the instrument computer before 3730/3730xl
Data Collection software is installed.
• Do not rename the computer once 3730/3730xl Data Collection software has been installed. Doing so will cause the 3730/3730xl Data Collection software to malfunction.
File-Naming
Convention
Some alphanumeric characters are not valid for user names or file names. The invalid characters are below: spaces
\ / : * ? " < > |
IMPORTANT!
An error message is displayed if you use any of these characters. You must remove the invalid character to continue.
What Plate
Records Contain
Plate records are data tables in the instrument database that store information about the plates and the samples they contain. Specifically, a plate record contains the following information:
• Plate name, type, and owner
• Position of the sample on the plate (well number)
• Sample name, see page page 74
• Mobility file (in Analysis Protocol), see page page 66
• Comments about the plate and about individual samples
• Name of the run module and Dye set information (run modules specify information
about how samples are run) (in Instrument Protocol), see page 62
• Name of the Analysis Protocol—Analysis Protocols specify how data is analyzed at the end of the run (in Analysis Protocol), see page
When to Create a
Plate Record
A plate record must be created for each plate of samples for the following types of runs:
• Spectral calibrations
• Sequencing analysis
• SeqScape analysis
Note:
A plate record must be created in advance of the first run. Plate records can be created, and plates added to the stacker, while a run is in progress.
Notes
58
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Plate Records and Sequencing Analysis
Sequencing
Analysis Plate
Record
The Plate Editor displays an empty plate record for the selected application that is chosen in the New Plate dialog box. The data fields within a given plate record vary depending on the selected application. This section describes the data fields that are present in a sequencing analysis Plate Record.
The table below and the flow chart on
page 59 describes what each file specifies:
Parameters Description
Instrument Protocol Contains everything needed to run the instrument.
Analysis Protocol Contains everything needed to analyze sequencing data.
Results Group Defines the file type, the file name, file save locations, analysis software and autoanalysis.
See Page
Plate Manager
Plate Record
Results Group Instrument Protocol Analysis Protocol
Default analysis protocols
File Save preferences
Run module
Dye set
Mobility
Bases called
(pure or mixed)
Post classification
(SQVs)
Post analysis processing
(clear range)
Elements of a Sequencing Analysis plate record
IMPORTANT!
In order for data collection and autoanalysis to be successful, each run of samples must have an Instrument Protocol, an Analysis Protocol, and a Results Group assigned within a plate record.
4
Notes
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
59
Chapter 4 Setting Up the Software for DNA Sequencing
Plate Records and Sequencing Analysis
1
2
3 4 5
Column
1. Sample Name
2. Comment
Default is one sample run. To add additional runs, see
Blank Sequencing Analysis plate record
The following table describes the columns inserted in a Plate Record for a sequencing analysis run.
Description
Name of the sample
Comments about the sample (optional)
• New: Opens the Results Group Editor dialog box
• Edit: Opens the Results Group Editor dialog box for the Results Group listed in the cell
• None: Sets the cell to have no selected Results Group
• Select one of the available Results groups from the list
Note:
You must have a Results Group selected for each sample entered in the Sample Name column.
See,
.
Notes
60
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Plate Records and Sequencing Analysis
Column Description
4. Instrument Protocol • New: Opens the Protocol Editor dialog box.
• Edit: Opens the Protocol Editor dialog box for the Instrument Protocol listed in the cell.
• None: Sets the cell to have no selected protocol.
• List of Instrument Protocols: In alpha-numeric order.
Note:
You must have an Instrument Protocol selected for each sample entered in the Sample
Name column.
See,
“Creating an Instrument Protocol” on page 62
.
5. Analysis Protocol • New: Opens the Analysis Protocol Editor dialog box.
• Edit: Opens the Analysis Protocol Editor dialog box for the Instrument Protocol listed in the cell.
• None: Sets the cell to have no selected protocol.
• List of Analysis Protocols: In alpha-numeric order
Note:
You must have an Analysis Protocol selected for each sample entered in the Sample
Name column.
See,
“Creating an Analysis Protocol” on page 66 .
4
Notes
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61
Chapter 4 Setting Up the Software for DNA Sequencing
Creating Required Settings for Automated Sequencing Analysis
Creating Required Settings for Automated Sequencing
Analysis
If the Settings Already Exist
If the appropriate instrument protocol, analysis protocol, and results group have been created, proceed to
“Creating and Completing a Sequencing Analysis
.
Instrument Protocols
An instrument protocol contains all the settings necessary to run the instrument. An instrument protocol contains the protocol name, type of run, run module, and dye set.
Creating an Instrument Protocol
1.
In the Tree pane of the Data Collection Software, click
GA Instruments
> ga3730 > Protocol Manager.
Create instrument protocols here
Create analysis protocols here
Notes
62
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Creating Required Settings for Automated Sequencing Analysis
Creating an Instrument Protocol
2.
In the Instruments Protocols section, click
.
The Protocol Editor opens.
3.
Complete the Protocol Editor:
a.
b.
c.
Type a name for the protocol.
Type a description for the protocol
(optional).
Select Regular in the Type drop-list.
3a
3c
3d
3e d.
Using the information in the table below, select the correct run module for your run.
Note:
To customize a run module, see
Customizing Run Modules” on page 64
.
Run Module
Capillary Array
Length (cm)
Sequencing Run
XLRSeq50_POP7
LongSeq50_POP7
FastSeq50_POP7
StdSeq36_POP7
RapidSeq36_POP7
50
50
50
36
36
Extra long read
Long read
Fast read
Standard read
Rapid read
* Approximate run times assume oven temperature has reached run temperature
Approximate Run
Times* (min)
180
120
60
60
35
Notes
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4
Chapter 4 Setting Up the Software for DNA Sequencing
Creating Required Settings for Automated Sequencing Analysis
e.
Using the information in the table below, select the correct Dye Set for your run.
Dye Set
E_BigDyeV1
Z_BigDyeV3
Chemistry
ABI P
RISM
®
BigDye
®
v1.1 Terminator
ABI P
RISM
®
BigDye
®
v3.1 Terminator
f.
Click .
Tip: Customizing Run Modules
You can modify default run modules to suit your particular needs.
1. Click
GA Instruments >
instrument name >
ga3730 >
Module Manager
.
2. Click .
The Run Module Editor dialog box opens.
3. Complete the Run Module Editor dialog box: a. Enter a name for your new module.
b. In the Type drop-list, select the type of module
(Regular, Spatial or Spectral).
c. In the Template drop-list, select a template module as a basis for the new module.
Note:
You cannot edit a default module installed with
3730/3730xl Data Collection software.
d. Optional: Enter a description of your new run module.
3a
3b
3c
3d
3e
Notes
64
e. Change to the desired module parameters using the range for the allowable parameters.
f. Click OK.
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Creating Required Settings for Automated Sequencing Analysis
Editable Run Module Parameters
Editable Run Module Parameters
Parameter Name
Oven_Temperature
PreRun_Voltage
PreRun Time
Injection_Voltage
Injection_Time
First_ReadOut_time
Second_ReadOut_Time
Run_Voltage
Voltage_Number_Of_Steps
Voltage_Step_Interval
Voltage_Tolerance
Current_Stability
Ramp_Delay
Data_Delay
Run_Time
Range Comment
18-70 C
0-15 kV
1-1800 sec
0-15 kV
1-90 sec
100-16000 millisec
100-16000 millisec
Temperature setting for main oven throughout run.
Pre run voltage setting before sample injection.
Prerun voltage time.
Injection voltage setting for sample injection.
Sample injection time.
The interval of time for a data point to be produced.
First_ReadOut_time should be equal to Second_ReadOut_time.
The interval of time for a data point to be produced.
Second_ReadOut_time should be equal to First_ReadOut_time.
0-15 kV
0-100 steps
0-180 sec
0.1-6 kV
0-2000 microA
Final run voltage.
Number of voltage ramp steps to reach Run_Voltage. We recommend that you do not change this value unless advised otherwise by Applied Biosystems support personnel.
Dwell time at each voltage ramp step. We recommend that you do not change this value unless advised otherwise by Applied
Biosystems support personnel.
Maximum allowed voltage variation. We recommend that you do not change this value unless advised otherwise by Applied
Biosystems support personnel. If it goes beyond tolerance and shuts off, contact Applied Biosystems tech support.
Maximum allowed electrophoresis current variation. Current fluctuations above this value will be attributed to air bubbles in system and the voltage automatically turned off. We recommend that you do not change this value unless advised otherwise by
Applied Biosystems support personnel.
1-1800 sec Delay During Voltage Ramp. We recommend that you do not change this value unless advised otherwise by Applied Biosystems support personnel.
1-1800 sec Time from the start of separation to the start of sample data collection.
300-14000 sec Duration data is collected after Ramp_Delay.
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Creating Required Settings for Automated Sequencing Analysis
Analysis Protocols
An analysis protocol contains all the settings necessary for analysis and post processing:
• Protocol name – The name, description of the analysis protocol, and the sequence file formats to be used
• Basecalling settings – The basecaller, DyeSet file, and analysis stop point to be used
• Mixed Bases – Option: to use mixed base identification, and if so, define the percent value of the second highest to the highest peak
• Clear Range – The clear range to be used based on base positions, sample quality values, and/or number of ambiguities (Ns) present
Note:
If you created an appropriate analysis protocol in the Sequencing Analysis software, you can use it in data collection software.
IMPORTANT!
Do not delete an Analysis Protocol during a run while it is being used for that run.
Autoanalysis will not be performed if you do so.
Creating an Analysis Protocol
Refer to the Applied Biosystems DNA Sequencing
Analysis Software v5.1 User Guide (P/N 4346366), chapter 8 for more information regarding analysis protocols
1.
In the Analysis Protocol section of the Protocol
Manager, click .
If more than one analysis application is installed on the data collection computer, the Analysis
Applications dialog box opens.
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Creating Required Settings for Automated Sequencing Analysis
Creating an Analysis Protocol
2.
Select Sequencing Analysis, then click
The Analysis Protocol Editor dialog box opens.
.
3.
In the General tab:
a.
b.
Enter a unique name and description for the new protocol.
Select the appropriate Sequence File formats settings.
Option
Write .Seq File check box
Write Standard
Chromatogram
Format file
(.scf)
Write Phred
(.phd.1) File
If checked, the software creates…
a .seq file for printing the sequence as text file or for using the file in other software.
• ABI format is used with
Applied Biosystems software.
• FASTA format is used with other software
When selected, the software creates a .scf file that can be used with other software. When created, the .scf extension is not appended to the file name.
When selected and the KB basecaller is used, the software creates a .phd.1 file that can be used with other software.
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4.
Select the Basecalling tab.
a.
Select the appropriate basecaller and
DyeSet primer based on the chemistry and capillary array length you are using.
Note:
Sequencing Analysis Software v5.1 and 3730/3730xl Data Collection software filter .mob file choices to match the chosen
.bcp file.
b.
In the Processed Data pane, select True or
Flat Profile.
Option Function
Used to display data as processed traces scaled uniformly so that the average height of peaks in the region of strongest signal is about equal to a fixed value.
The profile of the processed traces will be very similar to that of the raw traces.
Used to display the data as processed traces scaled semi-locally so that the average height of peaks in any region is about equal to a fixed value. The profile of the processed traces will be flat on an intermediate scale (> about 40 bases).
Note:
This option is applied to data that is analyzed with the KB basecaller only.
If you use the ABI basecaller the profile option reverts to True Profile.
c.
If desired, select one or more stop points for data analysis.
d.
Select your Threshold Quality option.
Option Function
When using the KB basecaller, use this option to assign a base to every position, as well as the QV.
When using the KB basecaller, use this option to assign Ns to bases with QVs less than the set point. The QV will still be displayed.
5.
Select the Mixed Bases tab.
Note:
This function is active with the KB
Basecaller only.
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4a
4c
4d
4b
Creating Required Settings for Automated Sequencing Analysis
Creating an Analysis Protocol
a.
b.
For mixed bases only, select Use Mixed
Base Identification.
Use the default setting of 25% or change the detection level by entering a new value or dragging the % line up or down.
Note:
Do not use less than 15% as your detection limit.
5a
5b
6.
Select the Clear Range tab.
Note:
The clear range is the region of sequence that remains after excluding the low-quality or error prone sequence at both the 5´ and 3´ ends.
Select one or more Clear Range methods. If you apply multiple methods, the smallest clear range results.
7.
Click to save the protocol and close the
Sequence Analysis Protocol Editor dialog box.
Use with ABI and
KB Basecallers
Use with
KB Basecaller
Use with ABI and
KB Basecallers
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Chapter 4 Setting Up the Software for DNA Sequencing
Creating Required Settings for Automated Sequencing Analysis
Editing and Deleting Analysis Protocols
Editing an Analysis Protocol
1.
In the Analysis Protocols pane in the Analysis
Protocol Manager, highlight the protocol you want to edit.
2.
Click .
3.
Make changes in the General, Basecalling,
Mixed Bases and Clear Range tabs, as appropriate.
4.
Click to save the protocol and close the
Analysis Protocol Editor dialog box.
Deleting an Analysis Protocol
IMPORTANT!
Do not delete an Analysis Protocol during a run while it is being used for that run.
Autoanalysis will not be performed if you do so. Also,
You must first delete any plate records using the
Analysis Protocol before you can delete or modify the
Analysis Protocol for these plate records.
1.
In the Analysis Protocols pane in the Analysis
Protocol Manager, highlight the protocol you want to delete.
2.
Click .
The Deletion Confirmation dialog box displays.
3.
Click .
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Exporting and Importing Analysis Protocols
Exporting and Importing Analysis
Protocols
Exporting an Analysis Protocol
1.
In the Analysis Protocols pane in the Analysis
Protocol Manager, highlight the protocol you want to export.
2.
Click .
The Export Confirmation dialog box displays.
3.
Click Save.
Importing an Analysis Protocol
1.
In the Analysis Protocols pane in the Analysis
Protocol Manager, highlight the protocol you want to import.
2.
Click .
The Export Confirmation dialog box displays.
3.
Click Save.
Results Groups
A Results Group is a component within Data
Collection that organizes samples and certain user settings under a single name. It is called a Results
Group because it is used to analyze, name, sort, and deliver samples that result from a run.
Creating a Results Group
1.
In the Tree pane of the Data Collection Software, click GA Instruments > Results Group.
2.
Click .
The Results Group Editor window displays.
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Creating Required Settings for Automated Sequencing Analysis
3.
Complete the General tab:
a.
b.
c.
Type a Results Group Name. The name can be used in naming and sorting sample files.
It must be unique (see page for a list of accepted characters).
Type a Results Group Owner (optional).
The owner name can be used in naming and sorting sample files.
Type a Results Group Comment (optional).
4.
Select the Analysis tab, then:
a.
b.
Select Sequencing Analysis from the
Analysis Type drop-list.
In the Analysis Actions section, select Do
Autoanalysis, if you want your data automatically analyzed after a run.
Note:
Login ID and password are not required for Sequencing Analysis software.
3a
3b
3c
4a
4b
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Creating Required Settings for Automated Sequencing Analysis
Creating a Results Group
5.
Select the Destination tab, then use the default destination or define a new location for data storage.
To use …
default location custom location
Then …
complete
and
5a a.
b.
Click Use Custom Location, then click location.
to navigate to a different save
Click to test the Location path name connection:
– If it passes, this text displays “Path Name test successful.”
– If it fails, this text displays “Could not make the connection. Please check that the Path Name is correct.” Click Browse and select a different location.
Sample File Destinations:
Locations where sample files are placed during extraction:
• Default Destination, default folder naming: Data / instrument type / instrument name / run folder (No ProcessedData folder)
• Default Destination, custom folder naming: Data/top custom folder/subfolders, etc.
• Custom Destination, default folder naming: Destination/instrument type/instrument name/run folder
• Custom Destination, custom folder naming: Destination/top custom folder/subfolders, etc.
5b
5c
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Chapter 4 Setting Up the Software for DNA Sequencing
Creating Required Settings for Automated Sequencing Analysis
6.
Select the Naming tab.
Use the Naming tab to customize sample file and run folder names.
IMPORTANT!
Sample name, run folder name, and path name, combined, can total no more than
250 characters. See page
for accepted characters.
The elements of the Naming tab are discussed in the following sections.
Sample File Name Format Pane
Follow the procedure below to complete the Sample
File Name Format pane.
1.
Select the Naming tab.
Sample
File Name
Format pane
Run Folder
Name
Format pane
2.
Click the Prefix box (optional) to type a prefix for the file name. Anything that you type here is shown in the Example line (see graphic below).
3.
Click the Name Delimiter list choose the symbol that will separate the Format elements in the file name (see step 3 below). Only one delimiter symbol may be chosen.
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Creating Required Settings for Automated Sequencing Analysis
Creating a Results Group
4.
Click the Format list and then select the components that you want in the sample name.
Note:
Generally, all the samples from a single run are placed in the same run or results folder, so the name of every sample from a single run should be different. Most of the Format options will not be different between samples, so you need to take care to select at least one of the options that make the sample names unique within a run.
For example, if a unique identifier is not included in the name, a warning message displays. The
Results Group makes the file name unique. As you select the elements for the file name, they are reflected in the Example line.
As you continue to select elements for the file name, additional elements display.
4
The names of the Format elements eventually truncate, but the Example field remains visible
(up to 72 characters).
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Creating Required Settings for Automated Sequencing Analysis
5.
Click the Suffix box (optional) and type the suffix for the file name.
The File Extension field displays the file extension generated from the Analysis Type specified on the Analysis tab (page
). For example, Sequencing Analysis produces sample files with an .ab1 extension.
Saving a Results Group
Click from any tab once all the elements within the Results Group have been chosen.
Note:
Even if you create a custom run folder location, a separate default run folder is generated that contains the log file.
Format Elements (Unique Identifiers)
While you may select a minimum of just one Format element for the Sample file and Run folder names in order to save a Results Group, selecting just the minimum may not provide enough information for you to identify the file or folder later.
Note:
If you choose a non-unique file name, the software appends numbers (incrementally) before the file extension.
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Creating Required Settings for Automated Sequencing Analysis
Format Elements (Unique Identifiers)
If you choose elements from the Format lists that do not create unique Sample file or
Run folder names, a warning message displays below the Example line (see next figure).
Warning message
To remove the warning message and proceed within the Results Group Editor window, simply select a Format element that distinguishes one file from another (for example, the capillary number is unique while the instrument name is not).
Run Folder/Sub-
Folder Name
Format Pane
Follow the same steps described above for the Sample File Name Format pane
(page
page 74 ) to specify the run folder name within the run folder.
Importing and
Exporting a
Results Group
Results Groups can be imported from, or exported to, tab-delimited text files. This allows easy sharing of identical Results Groups between instruments.
Importing a Results Group
1.
In the Tree pane of the Data Collection Software, click
GA Instruments > Results Group.
2.
Click .
A standard File Import dialog box displays.
3.
Navigate to the file you want to import.
Note:
Import file type is .txt (text).
4.
Click .
Note:
When you import or duplicate a Results Group, you are asked to type a name for the new Results Group and for the analysis application type.
Exporting a Results Group
1.
In the Tree pane of the Data Collection Software, click
GA Instruments > Results Group.
2.
Click the Results Group name to select it.
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Chapter 4 Setting Up the Software for DNA Sequencing
Creating Required Settings for Automated Sequencing Analysis
3.
Click .
A standard file export dialog box displays with the chosen Results Group name.
4.
Navigate to the location where you want to save the exported file.
5.
Click .
Note:
If there is a name conflict with a Results Group that already exists at the save location, the Results groups can be duplicated in order to copy settings into a similar Results Group without the risk of user error when copying it manually (see procedure below).
Duplicating a Results Group
1.
Click the Results Group to select it.
2.
Click .
Note:
When you import or duplicate a Results Group, you are asked to type a name for the new Results Group and for the analysis application type.
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Creating and Completing a Sequencing Analysis Plate Record
Format Elements (Unique Identifiers)
Creating and Completing a Sequencing Analysis Plate
Record
1.
In the Tree pane of the Data Collection Software, click GA Instruments > ga3730 >
Plate Manager.
2.
Click .
The New Plate Dialog dialog box opens.
3.
Complete the information in the New Plate
Dialog:
a.
b.
Type a plate ID or barcode.
Type a name for the plate.
c.
d.
e.
Type a description for the plate (optional).
Select your sequencing application in the
Application drop-list.
Select 96-well or 384-well in the Plate Type drop-list.
f.
Schedule the plate. For more information, see
“Scheduling Runs” on page 121 .
g.
Select heat seal or septa.
h.
Type a name for the owner and operator.
i.
Click .
The Sequencing Analysis Plate Editor opens.
3a
3b
3c
3i
3d
3e
3f
3g
3h
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Chapter 4 Setting Up the Software for DNA Sequencing
Creating and Completing a Sequencing Analysis Plate Record
Completing a Sequencing Analysis Plate
Record
Note:
Plate records can be imported and exported as tab-delimited files (.txt).
1.
In the Sample Name column of a row, enter a sample name, then click the next cell. The value
100 automatically display in the Priority column.
2.
In the Comments column, enter any additional comments or notations for the sample.
3.
In the Results Group 1 column, select a group
from the drop-list (see page 71 ).
4.
In the Instrument Protocol 1 column, select a protocol from the drop-list (see
).
5.
In the Analysis Protocol 1 column, select a protocol from the drop-list (see
).
4
1 2 3
5
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Creating and Completing a Sequencing Analysis Plate Record
Completing a Sequencing Analysis Plate Record
6.
To complete the rest of the plate record based on the samples loaded in your plate, do one of the following:
• For the same samples and protocols –
Highlight the entire row, then select Edit >
Fill Down Special (see
“Fill Down Special” on page 82
)
• Based on the plate type (96- or 384-well) and capillary array (48 or 96 capillaries) you are using, select the appropriate fill down option:
– 96 capillary/96-well plate: Fill Down.
– 48 capillary/96-well plate: Fill down
Special (48 Cap).
– 96 capillary/384-well plate: Fill down
Special (96 Cap).
– 48 capillary/384-well plate: Fill down
Special (48 Cap).
• For the same samples and protocols –
Highlight the entire row, then select Edit >
Fill Down.
• For the different samples and protocols, complete the plate editor manually.
7.
If you want to do more than one run, then select
Edit > Add Sample Run.
Additional Results Group, Analysis Protocol, and Instrument Protocol columns are added to the right end of the plate record.
You can add additional runs by selecting Edit >
Add Sample Run again.
8.
Complete the columns for the additional runs.
9.
Click .
IMPORTANT!
After clicking OK within the Plate
Editor, the completed plate record is stored in the
Plate Manager database. Once in the Plate
Manager database, the plate record can be searched for, edited, exported, or deleted.
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Fill Down Special
Fill Down Special
The following table illustrates the Fill Down Special feature.
If You Choose ...
Fill Down Special (48 Cap)
Then ...
The fill down pattern matches the 48-capillary load pattern.
First Quadrant
Second Quadrant
Fill Down Special (96 Cap)
*
The fill down pattern matches the 96-capillary load pattern.
*
Especially useful for 384-well plates
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Fill Down Special for a 48 Cap/96-well Plate
The Fill Down Special feature allows you to fill the plate record based on the load pattern of the capillary array that you are using.
To use the fill down special function:
1.
In the Plate Manager, double-click the plate of interest to display the Plate Editor.
2.
Type the sample name and then double-click it to highlight the entire row.
Fill Down Special
Completing a Sequencing Analysis Plate Record
4
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Chapter 4 Setting Up the Software for DNA Sequencing
Fill Down Special
3.
Select Edit > Fill Down Special (48 Cap) to fill the first quadrant.
4.
Click A02, type the name of sample 2 and highlight the entire row.
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5.
Select Edit > Fill Down Special (48 Cap) to fill the second quadrant.
Fill Down Special
Completing a Sequencing Analysis Plate Record
First
Quadrant
Fill Down Special for a 96 Cap/384-well Plate
this is how the fill down pattern looks when you use the Fill Down Special (96 Cap) feature on a 384-well plate.
Second
Quadrant
4
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Chapter 4 Setting Up the Software for DNA Sequencing
Fill Down Special
Adding a Sample Run
By adding additional sample runs, you can run samples with different variables
(different run modules, for example).
Select Edit > Add Sample Run
Adding an instance opens an additional:
• Results Group
• Instrument Protocol
• Analysis Protocol (sequencing only)
Notes
86
To run the plate(s), see “Running the Instrument” on page 115
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Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Setting Up the Software for
Fragment Analysis
Workflow
Preparing the
Instrument
Performing
Spatial Calibration
Performing
Spectral Calibration for Sequencing and
Fragment Analysis
Setting Up the Software for
DNA Sequencing
Setting Up the Software for
Fragment Analysis
Running the Instrument
Performing
Maintenance
Audit Trails and Access Control
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Create required settings for automated fragment analysis
Create and complete
a GeneMapper plate record
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3730/3730xl Data Collection and GeneMapper Software
3730/3730xl Data Collection and GeneMapper Software
Important Note
Do not rename the computer once 3730/3730xl Data Collection software has been installed. Doing so will cause the 3730/3730xl Data Collection software to malfunction.
File-Naming
Convention
Some alphanumeric characters are not valid for user names or file names. The invalid characters are below: spaces \ / : * ? " < > |
IMPORTANT!
An error message is displayed if you use any of these characters. You must remove the invalid character to continue.
Autoanalysis
You may choose to perform autoanalysis of fragment analysis samples by utilizing features of the 3730/3730xl Data Collection and GeneMapper software.
GeneMapper Software v3.5
Autoanalysis can be performed on the same instrument that collected the sample files or on a remote computer.
Manual Analysis
For information on manual analysis, refer to GeneMapper Software Version 3.5 User
Guide (PN 4343790)
Fragment
Analysis and Data
Collection
When GeneMapper software is installed on a computer that has 3730/3730xl DNA
Analyzer Data Collection Software, two applications are available through the Results
Group Editor (see page 102 ):
• GeneMapper-Generic and,
• GeneMapper-<Computer Name>
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3730/3730xl Data Collection and GeneMapper Software
GeneMapper-
Generic
GeneMapper-Generic enables you to generate .fsa files, but not perform autoanalysis.
When completing the Sample Sheet, you need to fill in basic information for Data
Collection to complete the run; all other GeneMapper software related fields are text entries. This is useful if you are using other software applications for analysis. This is also useful if you choose to analyze your samples in GeneMapper software on another computer, but do not have the same entries in the GeneMapper software database stored on the Data Collection computer. For example, if you have a customized size standard definition on the other GeneMapper software computer, you can type in that size standard name in the size standard text field and it will populate that column in your
GeneMapper software project.
GeneMapper-
<Computer
Name>
GeneMapper-<Computer Name> is for autoanalysis. The Size Standard, Analysis
Method, and Panel columns in the Sample Sheet window read directly from the
GeneMapper software database. These components must be created in GeneMapper software prior to setting up the plate record for a run. There is no way to create a new entry for these columns once inside the plate editor dialog box. If you create a new
GeneMapper software component while the plate record dialog box is open, the columns will not update. The plate record must be closed and reopened to update the
GeneMapper software components. For more information see, “Setting Up a Run for
Autoanalysis” on page 136 .
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Chapter 5 Setting Up the Software for Fragment Analysis
3730/3730xl Data Collection and GeneMapper Software
Notes
90
Workflow for
Autoanalysis
Using
GeneMapper
Software
Set up instrument and prepare samples
Is
GeneMapper registered and have user IDs been created?
Yes
Have you created definitions for
Size Std, Panel, and
Analysis Method in
GeneMapper software?
Yes
No
No
Do you have an
Instrument Protocol, and Results
Group?
No
Yes
Register software and create user IDs
Create definitions for:
Create
Instrument Protocol
Create
Results Group
Create and save plate record
Search for plate record, then schedule run
Open
Autoanalysis Manager
Start and monitor run
Autoanalysis Manager automatically processes the data
Review data in
GeneMapper software
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
GeneMapper Plate Records
GeneMapper Plate Records
Overview
A plate record is similar to a sample sheet or an injection list that you may have used with other Applied Biosystems instruments.
Plate records are data tables in the instrument database that store information about the plates and the samples they contain. Specifically, a plate record contains the following information:
• Plate name, type, and owner
• Position of the sample on the plate (well number)
• Comments about the plate and about individual samples
• Dye set information (in Instrument protocol)
• Name of the run module (run modules specify information about how samples are run) (in Instrument protocol)
When to Create a
Plate Record
A plate record must be created for each plate of samples for the following types of runs:
• Spectral calibrations
• Fragment analysis
Note:
A plate record must be created in advance of the first run. Plate records can be created, and plates added to the stacker, while a run is in progress.
Parameters
Instrument
Protocol
Results Group
Description
Contains everything needed to run the instrument.
See
Page
97
Defines the file type, the file name, autoanalysis, and file save locations that are linked to sample injections.
102
IMPORTANT!
In order for data collection and auto-analysis to be successful, each run of samples must have an Instrument Protocol and a Results Group assigned within a plate record.
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Elements of a GeneMapper Software Plate Record
Elements of a GeneMapper Software Plate Record
Plate Manager
Plate Record
Data Collection software files:
Results Group
Results Group
Name
Analysis software and autoanalysis
File storage location
File and run folder name preferences
Instrument Protocol
Instrument
Protocol Name
Run type
(Regular)
Run module
Dye set
GeneMapper software definitions:
Size standard
Analysis method
Bin set
Panel
SNP Set
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1 2
3 4 5 6
Elements of a GeneMapper Software Plate Record
7 8 9
10
Default is one sample run. To add additional runs, see page 112.
The following table describes the columns inserted in a Plate Record for a fragment analysis run.
1. Sample Name
2. Comment
3. Sample Type
Column
4. Size Standard
IMPORTANT!
For GeneMapper-<Computer Name> ONLY:
Size Standard, Panel, and Analysis Method must be created in GeneMapper software before creating a new plate
Description
Name of the sample
Comments about the sample (optional)
Use to identify the sample as Sample, Positive Control, Allelic
Ladder or Negative Control.
• GeneMapper-Generic (optional):
Manually enter size standards in the text field
• GeneMapper-<Computer Name>:
Select a saved size standard from the drop-list
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Elements of a GeneMapper Software Plate Record
Column
5. Panel
IMPORTANT!
For
GeneMapper-<Computer Name> ONLY:
Size Standard, Panel, and Analysis Method must be created in GeneMapper software before creating a new plate
6. Analysis Method
IMPORTANT!
For GeneMapper <Computer Name> ONLY:
Size Standard, Panel, and Analysis Method must be created in GeneMapper software before creating a new plate
7. Snp
IMPORTANT!
For
GeneMapper <Computer Name> ONLY:
Size Standard, Panel, and Analysis Method must be created in GeneMapper software before creating a new plate
8. 3 User-defined columns
Description
• GeneMapper-Generic (optional):
Manually enter panels in the text field*
• GeneMapper-<Computer Name>:
Select a saved panel from the drop-list
• GeneMapper-Generic (optional):
Manually enter analysis methods in the text field*
• GeneMapper-<Computer Name>:
Select a saved analysis method from the drop-list
• GeneMapper-Generic (optional):
Manually enter analysis methods in the text field*
GeneMapper-<Computer Name>:
Use for SNPlex chemistry; select a saved SNP set from the drop-list
Optional text entries
10. Instrument Protocol
• New: Opens the Results Group Editor dialog box
• Edit: Opens the Results Group Editor dialog box for the Results
Group listed in the cell
• None: Sets the cell to have no selected Results Group
• Select one of the available Results groups from the list
Note:
You must have a Results Group selected for each sample entered in the Sample Name column.
See, “Results Groups” on page 102 .
• New: Opens the Protocol Editor dialog box.
• Edit: Opens the Protocol Editor dialog box for the Instrument
Protocol listed in the cell.
• None: Sets the cell to have no selected protocol.
• List of Instrument Protocols: In alpha-numeric order.
Note:
You must have an Instrument Protocol selected for each sample entered in the Sample Name column.
• See, “Instrument Protocols” on page 97 .
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Creating Required Settings for Automated Fragment Analysis
If the Settings Already Exist
Creating Required Settings for Automated Fragment Analysis
If the Settings Already Exist
If the appropriate data collection and fragment analysis files have been created, proceed to “Creating and Completing a GeneMapper Plate Record” on page 110 .
Instrument Protocols
An instrument protocol contains all the settings necessary to run the instrument. An instrument protocol contains the protocol name, type of run, run module, and dye set.
Creating an Instrument Protocol
1.
In the Tree pane of the Data Collection Software, click GA Instruments > ga3730 >
Protocol Manager.
Create instrument protocols here
5
Create analysis protocols here
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Creating Required Settings for Automated Fragment Analysis
2.
In the Instruments Protocols section, click
.
The Protocol Editor opens.
3.
Complete the Protocol Editor:
a.
b.
c.
Type a name for the protocol.
Type a description for the protocol
(optional).
Select Regular in the Type drop-list.
3a
3b
3c
3d
3e d.
Select GeneMapper36_POP7.
e.
f.
Select G5.
Click .
Importing an Instrument Protocol
1.
Click in the Instrument Protocols pane of the Protocol Editor window.
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Creating Required Settings for Automated Fragment Analysis
Importing an Instrument Protocol
2.
Navigate to the protocol you want to import.
Note:
Import file type is .txt (text).
3.
Double-click the protocol to import it.
The imported files are displayed alphabetically in the Instrument Protocol pane.
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Chapter 5 Setting Up the Software for Fragment Analysis
Creating Required Settings for Automated Fragment Analysis
Customizing Run Modules
You can modify default run modules to suit your particular needs.
1. Click
GA Instruments > ga3730 >
Module
Manager.
2. Click .
3. Select a template module as a basis for the new module.
Choose module template from the drop-down menu (step 3).
4. Change to the desired module parameters using the table below as a guide to the allowable parameters.
Note:
You cannot edit a default module installed with 3730/3730xl Data Collection.
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The Run Module Parameters that you can edit:
Parameter Name
Oven_Temperature
PreRun_Voltage
PreRun Time
Injection_Voltage
Injection_Time
First_ReadOut_time
Second_ReadOut_Time
Run_Voltage
Voltage_Number_Of_Steps
Voltage_Step_Interval
Voltage_Tolerance
Current_Stability
Ramp_Delay
Data_Delay
Run_Time
Range Comment
18-70 C
0-15 kV
1-1800 sec
0-15 kV
1-90 sec
100-16000 millisec
100-16000 millisec
Temperature setting for main oven throughout run.
Pre run voltage setting before sample injection.
Prerun voltage time.
Injection voltage setting for sample injection.
Sample injection time.
The interval of time for a data point to be produced.
First_ReadOut_time should be equal to
Second_ReadOut_time.
The interval of time for a data point to be produced.
Second_ReadOut_time should be equal to
First_ReadOut_time.
Final run voltage.
0-15 kV
0-100 steps
0-180 sec
0.1-6 kV
Number of voltage ramp steps to reach Run_Voltage. We recommend that you do not change this value unless advised otherwise by Applied Biosystems support personnel.
Dwell time at each voltage ramp step. We recommend that you do not change this value unless advised otherwise by
Applied Biosystems support personnel.
Maximum allowed voltage variation. We recommend that you do not change this value unless advised otherwise by Applied
Biosystems support personnel. If it goes beyond tolerance and shuts off, contact Applied Biosystems tech support.
0-2000 microA
1-1800 sec
Maximum allowed electrophoresis current variation. Current fluctuations above this value will be attributed to air bubbles in system and the voltage automatically turned off. We recommend that you do not change this value unless advised otherwise by Applied Biosystems support personnel.
Delay During Voltage Ramp. We recommend that you do not change this value unless advised otherwise by Applied
Biosystems support personnel.
1-1800 sec Time from the start of separation to the start of data collection.
300-14000 sec Duration data is collected after Ramp_Delay.
5
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Chapter 5 Setting Up the Software for Fragment Analysis
Creating Required Settings for Automated Fragment Analysis
Results Groups
A Results Group is a component within Data
Collection that organizes samples and certain user settings under a single name. A Results Group is used to prepare samples for analysis and, to name, sort, and deliver samples that result from a run.
Creating a Results Group for Autoanalysis
1.
In the Tree pane of the Data Collection Software, click GA Instruments > Results Group.
2.
Click New.
The Results Group Editor window displays.
3.
Complete the General tab:
a.
b.
c.
Type a Results Group Name. The name can be used in naming and sorting sample files.
It must be unique (see page for a list of accepted characters).
Type a Results Group Owner (optional).
The owner name can be used in naming and sorting sample files.
Type a Results Group Comment (optional).
3a
3b
3c
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Creating Required Settings for Automated Fragment Analysis
Creating a Results Group for Autoanalysis
4.
Select the Analysis tab, then:
a.
Click the Analysis Type and then select one of the following:
If You Select ...
None
GeneMapper-
Generic
GeneMapper-
<Computer Name>
Then ...
Only raw data files are generated
Autoanalysis is not available and only .fsa files are generated
• Autoanalysis of completed runs is available
• Automated Processing tab is available
Steps b, c, and d below apply only to GeneMapper-
<Computer Name> (not
GeneMapper-Generic).
b.
In the Analysis Actions section, use the table below to select an option.
If You Select …
Do Autoanalysis
Do Autoanalysis and
Results Entry Group
Complete
Then …
Use with Setting from Automated
Processing Tab ( page 105 )
When every run completes
Samples are analyzed after each run of
48 or 96 samples.
Samples are analyzed after all samples using the same results group have been run.
Only when the result group is complete c.
d.
Type the Login ID.
Type the login password.
The login ID and password relate to the
GeneMapper software UserName and Password.
These items can only be created through the
GeneMapper software Options Users tab.
4a
4c
4d
4b
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Chapter 5 Setting Up the Software for Fragment Analysis
Creating Required Settings for Automated Fragment Analysis
5.
Select the Destination tab, then use the default destination or define a new location for data storage.
To use a …
default location custom location
Use for remote analysis using GeneMapper v3.5
Then …
skip to step 6 complete
and
below
a.
b.
Click Use Custom Location, then click location.
to navigate to a different save
Click to test the Location path name connection:
– If it passes, this text displays “Path Name test successful.”
– If it fails, this text displays “Could not make the connection. Please check that the Path Name is correct.” Click Browse and select a different location.
Sample File Destinations:
Locations where sample files are placed during extraction:
• Default Destination, default folder naming: Data / instrument type / instrument name / run folder (No ProcessedData folder)
• Default Destination, custom folder naming: Data/top custom folder/subfolders, etc.
• Custom Destination, default folder naming: Destination/instrument type/instrument name/run folder
• Custom Destination, custom folder naming: Destination/top custom folder/subfolders, etc.
5a
5b
5c
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Creating a Results Group for Autoanalysis
6.
Select the Naming tab.
Use the Naming tab to customize sample file and run folder names.
IMPORTANT!
Sample name, run folder name, and path name, combined, can total no more than
250 characters. See page 90 for accepted characters.
The elements of the Naming tab are discussed in the following sections, see page 106 .
Sample File Name Format pane
Run Folder Name Format pane
7.
Select the Automated Processing tab.
Note:
The Automated Processing tab is available only if you selected GeneMapper-
<Computer Name> in
In the “Autoanalysis is performed” section, use the table below to select when you want your samples autoanalyzed.
5
Select an autoanalysis option
8.
If You Select …
Only when the result group is complete
Samples are analyzed after all samples using the same results group have been run.
When every run completes
Then …
Samples are analyzed after each run of
48 or 96 samples.
Use with Settings from Analysis Tab
(
)
•
and
•
Do Autoanalysis and Results Entry
only
Click to save the Results Group.
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Chapter 5 Setting Up the Software for Fragment Analysis
Creating Required Settings for Automated Fragment Analysis
Sample File Name Format Pane
Follow the procedure below to complete the Sample
File Name Format pane.
1.
Click the Prefix box (optional) to type a prefix for the file name. Anything that you type here is shown in the Example line (see graphic below).
2.
Click the Name Delimiter list choose the symbol that will separate the Format elements in the file name (see step 3 below). Only one delimiter symbol may be chosen.
3.
Click the Format list and then select the components that you want in the sample name.
Note:
Generally, all the samples from a single run are placed in the same run or results folder, so the name of every sample from a single run should be different. Most of the Format options will not be different between samples, so you need to take care to select at least one of the options that make the sample names unique within a run.
For example, if a unique identifier is not included in the name, a warning message displays. The
Results Group makes the file name unique. As you select the elements for the file name, they are reflected in the Example line.
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Sample File Name Format Pane
As you continue to select elements for the file name, additional elements display.
Note:
An additional format list drop-menu displays after you select a format option.
The names of the Format elements eventually truncate, but the Example field remains visible (up to 72 characters).
Note:
To view the truncated format elements, place the cursor on the edge of the window until it turns into a double-arrow. Drag the arrow to expand the window horizontally.
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Chapter 5 Setting Up the Software for Fragment Analysis
Creating Required Settings for Automated Fragment Analysis
4.
Click the Suffix box (optional) and type the suffix for the file name.
The File Extension field displays the file extension generated from the Analysis
Type specified on the Analysis tab (
page 101 ). For example, fragment analysis
produces sample files with an .fsa extension.
Run Folder/Sub-Folder Name Format Pane
Follow the same steps described above for the Sample File Name Format pane
(
page 104 ) to change the sub-folder name within the run folder.
Format Elements (Unique Identifiers)
While you may select a minimum of just one Format element for the Sample file and
Run folder names in order to save a Results Group, selecting just the minimum may not provide enough information for you to identify the file or folder later.
Note:
If you choose a non-unique file name, the software appends numbers
(incrementally) before the file extension.
If you choose elements from the Format lists that do not create unique Sample file or
Run folder names, a warning message displays below the Example line
(see figure below).
Warning message
To remove the warning message and proceed within the Results Group Editor window, simply select a Format element that distinguishes one file from another (for example, the capillary number is unique while the instrument name is not).
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Creating Required Settings for Automated Fragment Analysis
Sample File Name Format Pane
Importing and
Exporting a
Results Group
Results Groups can be imported from, or exported to, tab-delimited text files. This allows easy sharing of identical Results Groups between instruments.
Importing a Results Group
1.
In the Tree pane of the Data Collection Software, click
GA Instruments > Results Group.
2.
Click .
A standard File Import dialog box displays.
3.
Navigate to the file you want to import.
Note:
Import file type is .txt (text).
4.
Click .
Note:
When you duplicate a Results Group, you are asked to type a name for the new Results Group and for the analysis application type.
Exporting a Results Group
1.
In the Tree pane of the Data Collection Software, click
GA Instruments > Results Group.
2.
Click the Results Group name to select it.
3.
Click .
A standard file export dialog box displays with the chosen Results Group name.
4.
Navigate to the location where you want to save the exported file.
5.
Click .
Note:
If there is a name conflict with a Results Group that already exists at the save location, the Results groups can be duplicated in order to copy settings into a similar Results Group without the risk of user error when copying it manually (see procedure below).
Duplicating a Results Group
1.
Click the Results Group to select it.
2.
Click .
Note:
When you duplicate a Results Group, you are asked to type a name for the new Results Group and for the analysis application type.
Notes
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Chapter 5 Setting Up the Software for Fragment Analysis
Creating and Completing a GeneMapper Plate Record
Creating and Completing a GeneMapper Plate Record
Creating the GeneMapper Plate Record for
Autoanalysis
1.
In the Tree pane of the Data Collection Software, click GA Instruments > ga3730 >
Plate Manager.
2.
Click .
The New Plate Dialog dialog box opens.
3.
Complete the information in the New Plate
Dialog:
a.
b.
Type a plate ID.
Type a name for the plate.
c.
d.
Type a description for the plate (optional).
Select your GeneMapper application in the
Application drop-list.
e.
f.
Select 96-well or 384-well in the Plate Type drop-list.
Schedule the plate. For more information, see
“Scheduling Runs” on page 121 .
g.
Select Heat Sealing or Septa.
h.
Type a name for the owner and the operator.
i.
Click .
The GeneMapper Plate Editor opens.
3d
3e
3f
3g
3h
3a
3b
3c
3i
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Creating and Completing a GeneMapper Plate Record
Completing a GeneMapper Plate Record for Autoanalysis
Completing a GeneMapper Plate Record for Autoanalysis
1.
In the Sample Name column of a row, enter a sample name, then click the next cell.
2.
In the Comment column, enter any additional comments or notations for the sample.
3.
In the Sample Type column, select a sample type from the drop-list.
4.
In the Size Standard column, select a size standard from the drop-list.
5.
In the Panel column, select a panel from the drop-list.
6.
In the Analysis Method column, select a method from the drop-list.
7.
In the Snp Set column, select a SNP set from the drop-list.
8.
Enter text for User-Defined columns 1 to 3.
9.
In the Results Group 1 column, select a group from the drop-list.
10.
In the Instrument Protocol 1 column, select a protocol from the drop-list.
4
1 2
5
8
6
3
9
7
10
5
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Chapter 5 Setting Up the Software for Fragment Analysis
Creating and Completing a GeneMapper Plate Record
11.
To complete the rest of the plate record based on the samples loaded in your plate, do one of the following:
• For the same samples and protocols –
Highlight the entire row, then select Edit >
Fill Down Special. For more information see,
“Fill Down Special” on page 111
.
• Based on the plate type (96- or 384-well) and capillary array (48 or 96 capillaries) you are using, select the appropriate fill down option:
– 96 capillary/96-well plate: Fill Down
– 48 capillary/96-well plate: Fill down
Special (48 Cap)
– 96 capillary/384-well plate: Fill down
Special (96 Cap)
– 48 capillary/384-well plate: Fill down
Special (48 Cap)
• For the different samples and protocols, complete the plate editor manually.
12.
If you want to do more than one run, then select
Edit > Add Sample Run.
Additional Results Group and Instrument
Protocol columns are added to the right end of the plate record.
You can add additional runs by selecting Edit >
Add Sample Run again (for more information see,
“Adding a Sample Run” on page 113
.
13.
Complete the columns for the additional runs.
14.
Click to save, then close the plate record.
IMPORTANT!
After clicking OK within the Plate
Editor, the completed plate record is stored in the
Plate Manager database. Once in the Plate
Manager database, the plate record can be searched for, edited, exported, or deleted.
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Fill Down Special
Completing a GeneMapper Plate Record for Autoanalysis
Fill Down Special
The following table illustrates the Fill Down Special feature.
If You Choose ...
Fill Down Special
(48 Cap)
Then ...
The fill down pattern matches the 48capillary load pattern.
First Quadrant
Second Quadrant
Fill Down Special
(96 Cap)
*
The fill down pattern matches the 96capillary load pattern.
5
*
Especially useful for
384-well plates
Fill Down Special for a 48 Cap/96-well Plate
The Fill Down Special feature allows you to fill the plate record based on the load pattern of the capillary array that you are using.
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Chapter 5 Setting Up the Software for Fragment Analysis
Fill Down Special
To use the fill down special function:
1. In the Plate Editor, complete the sample information in a row within the quadrant you want.
2. Highlight the entire row.
3. Select Edit > Fill Down Special (48 Cap) to fill the first quadrant.
4. Click A02, type the sample information, and highlight the entire row.
First
Quadrant
Second
Quadrant
5. Select Edit > Fill Down Special (48 Cap) to fill the second quadrant.
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Fill Down Special for a 96 Cap/384-well Plate
This is how the fill down pattern looks when you use the Fill Down Special (96 Cap) feature on a 384-well plate.
Fill Down Special
Adding a Sample Run
Adding a Sample Run
By adding additional sample runs, you can run samples with different variables (different run modules, for example).
Adding a sample run opens an additional:
• Results Group
• Instrument Protocol
1.
Select Edit > Add Sample Run
To Run the plate(s), see
“Running the Instrument” on page 115 .
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Chapter 5 Setting Up the Software for Fragment Analysis
Fill Down Special
Notes
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Running the Instrument
Workflow
Preparing the
Instrument
Performing
Spatial Calibration
Performing
Spectral Calibration for Sequencing and
Fragment Analysis
Setting Up the Software for
DNA Sequencing
Setting Up the Software for
Fragment Analysis
Running the Instrument
Performing
Maintenance
Audit Trails and Access Control
Notes
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Work with plate assemblies
Place plate assemblies into the instrument
Schedule a run
Run the instrument
6
Control the run
Work with data in the
Run History view
View analyzed data
115
Chapter 6 Running the Instrument
Working with Plate Assemblies
Working with Plate Assemblies
Plate Assembly
Components
Do not use warped or damaged plates.
Materials Required for Septa Assemblies
Each 96- or 384-well septa assembly contains a:
• Plate retainer
• Plate septa
• Sample plate
• Base plate
Use only black plate bases with septa-sealed plates.
96-well
Plate retainer
Plate septa
Sample plate
Black plate base
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Working with Plate Assemblies
Materials Required for Heat-sealed Assemblies
Each 96- or 384-well heat-sealed assembly contains a:
• Plate retainer
• Heat seal film
• Sample plate
• Base plate
Use only gray plate bases with heat-sealed plates.
Plate retainer
Heat seal film
sample plate
Gray plate base
Assembled components
384-well plate
IMPORTANT! Heat Seal Recommendations
• Use 3-mil Applied Biosystems heat seal film (PN 4337570). This film is 3-mil before, and 1-mil after, heating.
• Do not use heat seal film thicker than 1-mil, after heating, on 3730/3730xl DNA Analyzer.
• Do not use heat-seal film containing adhesives or metals as these may damage the instrument’s piercing needles
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6
Chapter 6 Running the Instrument
Working with Plate Assemblies
Preparing a Septa Plate Assembly
1.
Seal the plate:
a.
b.
Place the plate on a clean, level surface.
Lay the septa flat on the plate.
c.
Align the holes in the septa strip with the wells of the plate, then firmly press downward onto the plate.
2.
To prevent damage to the capillary array, inspect the plate and septa to verify the septa fits snugly and flush on the plate.
3.
Assemble the plate assembly:
a.
b.
Place the sample plate into the plate base.
Snap the plate retainer onto the plate and plate base.
Notes
118
Plate septa
Sample plate
Septa and well not aligned
Septa and well not aligned
3a
3b
Assembled components
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
4.
Verify that the holes of the plate retainer and the septa strip are aligned. If not, re-assemble the plate assembly (see
IMPORTANT!
Damage to the array tips will occur if the plate retainer and septa strip holes do not align correctly.
Working with Plate Assemblies
Preparing a Septa Plate Assembly
Plate retainer holes and septa holes are not aligned
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6
Chapter 6 Running the Instrument
Placing Plate Assemblies into the Instrument
Placing Plate Assemblies into the Instrument
1.
Open the stacker drawer.
2.
Open the door of the In Stack tower.
Stacker drawer
3.
Place the plate assemblies into the stacker in any order.
IMPORTANT!
When placing the plate into the stacker, the plate must be oriented so that the notched corner of the plate assembly is located in the rear-right corner of the stacker.
IMPORTANT!
in the stacker.
Do not place more than 16 plates
4.
Close the metal In Stack tower door.
5.
Close the Stacker drawer.
Notched corner of the plate assembly
≤ 16 plate assemblies
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Scheduling Runs
In the Tree pane of the Data Collection Software, click GA Instruments > ga3730 >
instrument name >
Run Scheduler.
Scheduling Runs
Preparing a Septa Plate Assembly
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Chapter 6 Running the Instrument
Scheduling Runs
384-Well Plate Mapping and Default Run
Scheduling
Samples within a plate run in the order of their well designation. For example, a default 384-well injection pattern looks like this:
• Plates that contain samples in a single quadrant and with more than one instrument protocol specified, run all the protocols in the order they appear in the plate record before the next quadrant is run.
Note:
The analysis module of a sample plays no part in the order in which that sample quadrant runs.
Quadrant 1: wells A1, C1, E1, G1...
Quadrant 2: wells B1, D1, F1, H1...
Quadrant 3: wells A2, C2, E2, G2...
Quadrant 4: wells B2, D2, F2, H2...
For information on setting up a Plate record, page 58
for sequencing, and
page 91 for fragment analysis.
The following table lists the default run priorities and load positions
Number of
Capillaries
96
48
Plate Size
384-well
96-well
3
4
1
2
1
Run
Priority
Quadrant First Load Position
Q1
Q2
Q3
Q4
Q1, load 1
Q1, load 2
Well A1
Well B1
Well A2
Well B2
Well A1
Well A2
48 384-well 1
2
3
4
Q1, load 1
Q1, load 2
Q2, load 1
Q2, load 2
Q3, load 1
Q3, load 2
Q4, load 1
Q4, load 2
Well A1
Well A3
Well B1
Well B3
Well A2
Well A4
Well B2
Well B4
Note:
When using a 384-well plate and a 48-capillary array, you can change the run order of the main quadrant (bold numbers above) but not the load numbers.
Globally Modifying a Run Schedule
You can change the run order of quadrants and then apply it to all 384-well plates.
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To modify the run order for all 384-well plates:
1.
Click your instrument name in the left pane.
2.
Select Instrument > Scheduling Preference.
The Default 384 well scheduling preference dialog box displays.
3.
Select the quadrant priority (run order) from the
Quadrant list.
You may select any run order. The example to the right shows a 4-3-2-1 quadrant priority (run order). With a 384-well and a 96-capillary array, the samples would run in this order:
B2, A2, B1, A1...
Scheduling Runs
Globally Modifying a Run Schedule
6
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Chapter 6 Running the Instrument
Scheduling Runs
Locally Modifying a Run Schedule
You can also change the run order of quadrants within a specific sample plate.
To locally modify the run order within a single
384-well plate:
1.
In the Plate Manager, click New Plate.
Note:
For information about the Plate Manager,
see page 79 for sequencing, and
fragment analysis.
2.
Select 384-Well from the Plate Type list.
The Scheduling box is activated.
3.
Type the run priority in the Scheduling box.
4.
Click OK.
Type run priorities here
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Default Load Maps
Default Load Maps
Refer to the following load maps for different sized arrays and sample plates.
96-Well Plate,
48 Capillaries
Sample Plate: 96-well, Array: 48-capillary
A
1
8
B
7
C
6
D
5
E
4
F
3
G
2
2
8
7
6
5
4
3
2
3
16
15 15 23 23 31 31 39 39 47
47
H
1 1
= First load
= Second load
9
4
16
5
24
6
24
7
32
8
32
9
40
14 14 22 22 30 30 38
13 13 21 21 29 29 37
12 12 20 20 28 28 36
11 11 19 19 27 27 35
10 10 18 18 26 26 34
9 17 17 25 25 33
10
40
11
48
12
48
38 46 46
37 45 45
36
35
44
43
44
43
34 42 42
33 41 41
GR2220 well number capillary number
96-Well Plate,
96 Capillaries
Sample Plate: 96-well, Array: 96-capillary
A
1
15
B
13
2
16
3
30
4
32
14 29 30
C
11 12 27 28
5
47
6
48
7
63
45 46 61
8
64
9
79
10
80
62 77 78
10 25 26
43 44 59
41 42 57
60 75 76
58 73 74
D
9
E
7
F
5
G
3
H
1
8
6
4
2
23 24 39 40 55
21 22
19 20
56 71 72 87 88
37 38 53 54 69 70
35 36 51 52 67 68
17 18 33 34 49
11
95
12
96
93 94
91 92
89 90
85 86
83 84
50 65 66 81 82
GR2219 well number capillary number
6
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Chapter 6 Running the Instrument
Default Load Maps
384-Well Plate, 48
Capillaries
First quadrant pickup
G
H
I
E
F
J
K
L
M
N
O
P
A
B
C
D
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
8 8 16 16 24 24 32 32 40 40 48 48
7
6
5
4
3
2
1
7
6
5
4
3
2
1
15
14
13
12
11
10
9
15
14
13
12
11
10
9
23
22
21
20
19
18
17
23
22
21
20
19
18
17
31
30
29
28
27
26
25
31
30
29
28
27
26
25
39
38
37
36
35
34
33
39
38
37
36
35
34
33
47
46
45
44
43
42
41
47
46
45
44
43
42
41
GR2222a
= First load
= Second load
Second quadrant pickup well number
Capillary number
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
D
E
F
G
H
I
J
K
A
B
C
L
M
N
O
P
8
7
6
5
4
3
2
1
8
7
6
5
4
3
2
1
16
15
14
13
12
11
10
9
16
15
14
13
12
11
10
9
24
23
22
21
20
19
18
17
24
23
22
21
20
19
18
17
32
31
30
29
28
27
26
25
32
31
30
29
28
27
26
25
40
39
38
37
36
35
34
33
40
39
38
37
36
35
34
33
48
47
46
45
44
43
42
41
48
47
46
45
44
43
42
41
GR2222b
= First load
= Second load
Third quadrant pickup
L
M
I
J
K
N
O
P
A
B
C
D
E
F
G
H
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
8 8 16 16 24 24 32 32 40 40 48 48
7
6
5
4
3
2
1
7
6
5
4
3
2
1
15
14
13
12
11
10
9
15
14
13
12
11
10
9
23
22
21
20
19
18
17
23
22
21
20
19
18
17
31
30
29
28
27
26
25
31
30
29
28
27
26
25
39
38
37
36
35
34
33
39
38
37
36
35
34
33
47
46
45
44
43
42
41
47
46
45
44
43
42
41
GR2222c
= First load
= Second load
Fourth quadrant pickup well number
Capillary number
H
I
J
K
E
F
G
A
B
C
D
L
M
N
O
P
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
8
7
6
5
4
3
2
1
8
7
6
5
4
3
2
1
16
15
14
13
12
11
10
9
16
15
14
13
12
11
10
9
24
23
22
21
20
19
18
17
24
23
22
21
20
19
18
17
32
31
30
29
28
27
26
25
32
31
30
29
28
27
26
25
40
39
38
37
36
35
34
33
40
39
38
37
36
35
34
33
48
47
46
45
44
43
42
48
47
46
45
44
43
42
41 41
GR2222d
= First load
= Second load
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Default Load Maps
384-Well Plate, 96
Capillaries
First quadrant pickup
G
H
I
L
M
J
K
N
O
P
C
D
E
F
A
B
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
15 16 31 32 47 48 63 64 79 80 95 96
13
11
14
12
29
27
30
28
45
43
46
44
61
59
62
60
77
75
78
76
93
91
94
92
9
7
5
3
1
10
8
6
4
2
25
23
21
19
17
26
24
22
20
18
41
39
37
35
33
42
40
38
36
34
57
55
53
51
49
58
56
54
52
50
73
71
69
67
65
74
72
70
68
66
89
87
85
83
81
90
88
86
84
82
GR2221a
Well number
Capillary number
Third quadrant pickup
G
H
I
L
M
J
K
N
O
P
C
D
E
F
A
B
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
15 16 31 32 47 48 63 64 79 80 95 96
13
11
9
7
5
3
1
14
12
10
8
6
4
2
29
27
25
23
21
19
17
30
28
26
24
22
20
18
45
43
41
39
37
35
33
46
44
42
40
38
36
34
61
59
57
55
53
51
49
62
60
58
56
54
52
50
77
75
73
71
69
67
65
78
76
74
72
70
68
66
93
91
89
87
85
83
81
94
92
90
88
86
84
82
GR2221c
Second quadrant pickup
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
C
D
E
A
B
15
13
J
K
L
M
N
O
P
H
I
F
G
11
9
7
5
3
1
16
14
12
10
8
6
4
2
31
29
27
25
23
21
19
17
32
30
28
26
24
22
20
18
47
45
43
41
39
37
35
33
48
46
44
42
40
38
36
34
63
61
59
57
55
53
51
49
64
62
60
58
56
54
52
50
79
77
75
73
71
69
67
65
80
78
76
74
72
70
68
66
95
93
91
89
87
85
83
81
96
94
92
90
88
86
84
82
GR2221b
Fourth quadrant pickup
F
G
H
I
J
K
L
M
N
O
P
C
D
E
A
B
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
15
13
11
9
7
5
3
1
16
14
12
10
8
6
4
2
31
29
27
25
23
21
19
17
32
30
28
26
24
22
20
18
47
45
43
41
39
37
35
33
48
46
44
42
40
38
36
34
63
61
59
57
55
53
51
49
64
62
60
58
56
54
52
50
79
77
75
73
71
69
67
65
80
78
76
74
72
70
68
66
95
93
91
89
87
85
83
96
94
92
90
88
86
84
81 82
GR2221d
For a 384-well plate, injections are made from every other well and every other row. A full 384-well plate requires 4 runs for a 96-capillary array, and 8 runs for 48-capillary array, to inject all the samples once.
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Chapter 6 Running the Instrument
Barcode Readers
Barcode Readers
Internal Barcode
Reader
ELECTRICAL HAZARD. Power off the instrument and the computer before connecting an external barcode reader to the instrument.
The 3730 & 3730xl internal barcode reader supports the following formats:
• Code 128
• Code 39
• Code 93
• LOGMARS
• EAN-8
Note:
All Applied Biosystems barcoded plates for the 3730 and 3730xl instruments are code 128 format.
The barcode reader shares the default Windows illegal character list, which means the illegal characters for the reader are \ / : * ? " < > | and also having a space is illegal.
External Barcode
Readers
KEYENCE BL-80VE
An external barcode reader can also be used with the 3730 and 3730xl instruments. We have experience with the KEYENCE BL-80VE (see photo above), which connects to the instrument computer keyboard. With this reader, you can scan barcodes into any text box in the Data Collection software.
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Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Barcode Readers
ELECTRICAL HAZARD. Power off the instrument and the computer before connecting an external barcode reader to the instrument.
KEYENCE 80RKE
Another option is the KEYENCE 80RKE which you connect to the instrument serial port. With this reader, you can scan barcode information only into specific text boxes within the Data Collection software.
Note:
The 80RE is not supported for either instrument.
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Chapter 6 Running the Instrument
Running the Instrument: Manual vs Auto Mode
Running the Instrument: Manual vs Auto Mode
Accessing Modes
You may schedule a run or runs using either manual mode or auto mode. Both modes are described below.
Access either mode by selecting:
Run Scheduler >Instrument > Instrument Name >
Run mode (Auto or Manual)
Note:
You must be in the Run Scheduler view to see the instrument run mode menu.
Manual Mode Features
The benefits and features of using manual mode are:
• Plates can be added to the stacker individually and in order; runs are scheduled in the order the plates are in the stack.
• The internal reader is not necessary to link plates to plate records in the local database.
• Plates do not need to have a barcode.
Scheduling Runs Using Manual Mode
To schedule runs using the manual mode (default):
1.
Click the Run Scheduler icon.
2.
Select Instrument > Instrument Name >
Manual mode.
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3.
Click Search in the Run Scheduler to search for plate record(s).
Click Search
Up and Down buttons
Running the Instrument: Manual vs Auto Mode
Scheduling Runs Using Manual Mode
This opens the Add Plates to In Stack dialog box.
4.
Type the name of the plate(s) or scan the plate ID and click Search.
Barcode search
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Advanced search
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Chapter 6 Running the Instrument
Running the Instrument: Manual vs Auto Mode
5.
Select run(s) to add and then click Add to add the plate record(s) to the Input Stack in the order in which you want them to run.
6.
Click Done to close the Add Plates to In Stack dialog box.
7.
Physically stack the plates in the In Stack in order. The bottom plate runs first.
IMPORTANT!
The order of the plate record must match the stack order of the plates in the In
Stack. If the order does not match, processed runs will have the wrong plate record information.
Note:
You may assign more plates in the Run
Scheduler than are actually available in the stacker.
8.
Click (Run).
As the plates are retrieved by the autosampler, they are run in the order they were placed in the
In Stack.
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Auto Mode Features
The features and benefits of the using Auto Mode are:
• Plates must have barcodes.
• Internal barcode reader is necessary in order to link plates to plate records in the local database.
• You can add plates to the In Stack in any order.
• Plates can be added or removed during instrument operation.
To schedule runs using the Auto mode:
1.
Select Run Scheduler > Instrument Name >
Auto mode.
Notice that the Search, Up, and Down buttons are no longer visible as they are in Manual mode.
Also, you no longer have the Add Plate (Scan or
Type Plate ID) option as you do in Manual mode.
2.
Physically place plates in the In Stack in any order. Remember that the bottom plate runs first, the top plate runs last.
3.
Click
As the plates are retrieved by the autosampler, plate barcodes are scanned and their plate records are associated with those stored in the local data collection database.
Running the Instrument: Manual vs Auto Mode
Auto Mode Features
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Chapter 6 Running the Instrument
Running the Instrument: Launching the Run
Running the Instrument: Launching the Run
1.
Verify the active spectral calibration matches your dye set and capillary array length.
2.
If you want to review the run schedule before beginning the run, click
GA Instruments > ga3730 >
instrument name >
Run Scheduler
3.
Click the green button in the toolbar.
The Processing Plates dialog box opens.
4.
Click OK.
5.
The software automatically checks:
– the capillary array length and polymer type in the Instrument Protocol column of the plate record against the capillary array length and polymer type
– the available space in the database and drive E
If the database or drive E are …
full not full
Then …
A warning displays. Do the following:
1. Make more space by deleting unneeded files. See,
“Working With Drives for Database and
Sample Data Storage” on page 174
2. Click the green button to start the run.
the run starts.
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Running the Instrument: Launching the Run
Basic Run Module Steps
Basic Run Module Steps
When the run starts, the following basic steps are performed automatically by the instrument
Module Steps Approximate Time
Turn Oven On N/A
Wait for oven to equilibrate
Initialize autosampler
Fill Array
PreRun
Inject samples
Start separation
Ramp voltage
1 min 40 sec (when oven is at set temperature)
3-4 min
3 min
30 sec
10 min
Collect Data
Run ends:
Leave oven on
Laser to idle
Variable
Until next run starts
Total time prior to separation:
• Cold start: ~38 minutes
• Warm start ~10 minutes (oven is already at temperature)
Note:
A PostBatch Utility, which runs automatically, turns off the oven and the laser at end of a batch of runs.
DNA Sequencing Run Times
The following table lists the approximate run times of common DNA sequencing analysis runs:
Analysis Capillary Array Length
Rapid read DNA sequencing 36-cm
Standard read DNA sequencing
Fast DNA sequencing
36-cm
50-cm
Long read DNA sequencing
Extra Long DNA sequencing a. Times assume oven is at temperature
50-cm
50-cm
Run Module
RapidSeq36_POP7
StdSeq36_POP7
FastSeq50_POP7
LongSeq50_POP7
XLRSeq50_POP7
Approximate
Run Time a
(min)
35
60
60
120
180
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Chapter 6 Running the Instrument
Running the Instrument: Launching the Run
Fragment Analysis Run Times
The following table lists the approximate run time of a common fragment analysis run:
Capillary Array Length
36-cm
Run Module
GeneMapper36_POP7
Instrument Status Lights
Status
Status
Light
• The instrument is ready.
• An automated wizard operation is in progress with the instrument door closed.
• A run is in progess
Solid green
Flashing green
Action
Go to
• The instrument is downloading firmware.
Flashing yellow
• The instrument cannot communicate with the computer.
• The instrument has detected a problem.
Solid yellow
• The instrument is performing diagnostics.
Flashing yellow
• The oven door is open.
• The instrument door is open.
• The buffer reservoir is not installed.
• The capillary array is not installed.
• An automated wizard operation is in progress with the instrument door open.
Solid red
Go to
Go to
Go to
Approximate
Run Time (min)
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Controlling the Run
Fragment Analysis Run Times
Controlling the Run
You can use the toolbar at the top of the data collection software window to control the run.
To ...
Start the run
Stop the current run
Stop after the current run
Skip to next run
Pause after current run
Resume after pause
Click ...
Starts run(s).
Action
Stops the current run.
Finishes current run and then stops.
Stops the current run and begins next scheduled run.
Finishes current run and then waits for resume command to begin next scheduled run.
Begin the next scheduled run after a pause.
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Chapter 6 Running the Instrument
Controlling the Run: Instrument Status
Controlling the Run: Instrument Status
Click (Instrument Status) to monitor the status of the instrument or the current run.
System Status must be
‘Ready’ before a run starts
Array and polymer information
System Status changes from green to flashing red when errors occur.
Events Box
The Events box lists the:
• Instrument’s recent actions
• Status of each capillary as passed or failed at the end of a spectral calibration
• Calibration data at the end of a spatial calibration
Some of the events listed in the Events box provide information for service engineers.
Errors Box
The Errors box lists errors that have occurred during the current run.
Some of the error messages provide information for service engineers. A “fatal” error usually requires that you restart the data collection software.
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Controlling the Run: EPT Chart
Fragment Analysis Run Times
Controlling the Run: EPT Chart
The EPT Viewer displays real-time electrophoresis (EP) data during a run.
In the tree pane of the Data Collection Software, click GA Instruments >
ga3730 > instrument name > Instrument Status > EPT Chart.
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Chapter 6 Running the Instrument
Controlling the Run: Event Log
Controlling the Run: Event Log
The Event log graphically itemizes events such as errors and general information, as the graphic below illustrates.
In the tree pane of the Data Collection Software, click GA Instruments >
ga3730 > instrument name > Instrument Status > Event Log. This opens the event log window.
Clear error messages by clicking Clear Errors. The System Status light flashes red until all errors are cleared.
Note:
This view can also be used to monitor a spectral calibration run in real time to verify the capillary-by-capillary processing status.
Notes
140
Note:
If an error is generated while using manual control, re-boot the instrument and
Data Collection software to recover from the error stage.
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Controlling the Run: Capillary Viewer
Fragment Analysis Run Times
Controlling the Run: Capillary Viewer
Viewing Data in the Capillary
Viewer
Use the Capillary Viewer to examine the quality of electropherogram data during a run for several capillaries at once.
In the tree pane of the Data Collection Software, click GA Instruments >
ga3730 > instrument name > Instrument Status > Capillary Viewer.
Electropherogram Displays
An electropherogram is a graph of relative dye concentration against time, plotted for each dye. The data displayed has been corrected for spectral overlap
(multicomponented).
How to Zoom
To zoom:
1.
Hold and drag the mouse over the area of interest.
2.
Release the mouse, then click to expand the view.
3.
Click to return to full view.
Click individual colors to view or hide them.
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Chapter 6 Running the Instrument
Controlling the Run: Capillary Viewer
Viewing Data in the Array Viewer
Use this during or after a run to examine the quality of your data, which is displayed as color data for the entire capillary array. You can view all the capillaries (vertical axis) as a function of time/data point (horizontal axis).
In the tree pane of the Data Collection Software, click GA Instruments >
ga3730 > instrument name > Array Viewer. This opens the Array Viewer window.
Capillary 1
Notes
142
Capillary 96 or
Capillary 48
How to Zoom 1.
To expand the view, hold and drag the mouse over the area of interest.
2.
Click to return to full view.
Color Bar
Click individual colors to view or hide them (same in Capillary Viewer).
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Working with Data in The Run History View
Color Bar
Working with Data in The Run History View
Run History
Components
The Run History utility can be used only with completed runs stored in the local 3730
Data Collection database. It does not provide real-time viewing of collecting runs.
In the left tree pane, click the icon next to the function to launch it.
Icon Elements Within the Run History Utility
EPT Viewer
Note:
If Cleanup Database has been used, you cannot view processed data in Run History.
Spatial Calibration Viewer
Capillary Viewer
Note:
If Cleanup Database has been used, you cannot view processed data in Run History.
Array Viewer
Note:
If Cleanup Database has been used, you cannot view processed data in Run History.
Spectral Calibration Viewer
Reextraction
Note:
If Cleanup Database has been used, you cannot view processed data in Run History.
Viewing Data from a Completed Run in the
Data Collection
Software
There are two formats for viewing data within the 3730 Data Collection Software under the Run History icon:
• In the Array Viewer window
• In the Capillary Viewer window, capillary-by-capillary
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Chapter 6 Running the Instrument
Working with Data in The Run History View
Viewing Data from a Completed Run
1.
On the left tree pane in the 3730/3730xl Data Collection software, click
(Run History) to select the run you want to view.
2.
Search for your run by either Barcode or Advanced search.
3.
After choosing the run, click the Array Viewer or the Capillary Viewer from the left tree pane.
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Viewing the Results of Autoextraction
Color Bar
Viewing the Results of Autoextraction
After a run is completed, extraction and analysis is performed automatically, according to the settings in the Plate Editor and the Results Group. The results of extraction and analysis can be viewed in the Reextraction Panel. Samples can be extracted again with the same settings, or with different Analysis Protocols or different Results Groups. This can be useful for several reasons:
• The destination location may not have been available during extraction.
• Some samples may have failed analysis and a different Analysis Protocol might be more successful.
• Samples might be saved in different locations, or with no analysis at all to save space.
• Sample files are created based on the your destination and folder naming selections.
Runs Stopped
Before Complete
Autoextraction
Runs that are stopped before completion display the status “Completed” in the Run
Scheduler and the plate is moved to the Out Stack. In the Instrument View the status is changed to “Ready.” Successfully extracted and analyzed runs display the status
“Processed” in the Run Scheduler.
The auto extractor component of the 3730/3730xl Data Collection automatically extracts data from stopped runs. If autoextraction fails, click the Reextraction icon to extract data.
Effects of
Changes Made in the Reextraction
Panel
Changes made in the Reextraction Panel to a Sample name, Analysis protocol, etc., also change in the original plate record. The original plate information is overwritten.
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Chapter 6 Running the Instrument
Viewing the Results of Autoextraction
Selecting and Queuing Samples for Reextraction
You can queue individual samples for reextraction. This is especially useful for experimenting with different Analysis Protocols for samples that have failed initial extraction.
1.
Click (Run History).
2.
Enter the plate ID for a plate that has been completed, or click Search. Plates that have runs still pending cannot be reextracted. All the runs from that plate appear in the window.
3.
Select a run from the list.
Notes
146
4.
Click (Reextraction) in the left tree pane.
The Reextraction window displays
5.
Click the checkboxes in the Extract column to select the samples to be reextracted.
6.
Click Extract to start the reextraction.
Note:
Reextracted sample files are saved in the original folder that data was extracted to, unless you modify the results group settings.
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Viewing the Results of Autoextraction
Elements of the Reextraction Window
Elements of the Reextraction Window
All the samples are displayed with the results of extraction and analysis.
Note:
Sort the columns of the re-extraction panel by holding the shift key and then clicking on a column header.
Reextraction Window for Sequencing
Analysis
Use check boxes to select samples to be reextracted
Select a run
Results of extraction and analysis
Click here to start extraction
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These are used if several samples are highlighted
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Chapter 6 Running the Instrument
Viewing the Results of Autoextraction
Reextraction Window for Fragment
Analysis
Use check boxes to select samples to be reextracted
Select a run
Results of extraction
Click here to start extraction
These are used if several samples are highlighted
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Viewing the Results of Autoextraction
Reextraction Window for Fragment Analysis
Results Column
The results of extraction and analysis are color coded in the Results column. The following table lists the colors and their values.
Color Value Notes
Red
Yellow *
Extraction or analysis failed
Warnings for extraction or analysis
Descriptive messages can be viewed by resizing the Results column to view all text
(click on the arrow)
Green Successful extraction (with no analysis intended), or successful extraction and analysis.
* Note: The text message for samples that produce yellow is: “FAILURE: Analysis Failed
Bad Data; Error Number=nnnnn
WARNING...
The Results column, by default, shows only the beginning of any processing message.
The entire message returned from extraction and autoanalysis is inside the cell and can be viewed by expanding the cell. The location of the stored sample is also found there. In addition, there is a tooltip view for each sample results message.
Tooltip view. Access by placing the cursor over the sample of interest
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Expanded column
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Chapter 6 Running the Instrument
Viewing the Results of Autoextraction
Quality Column
The Quality column represents the quality values for an entire sequence. Quality Values are only assigned to analyzed samples when using the KB Basecaller. The following table lists the displayed colors and their associated value range.
Color Quality Value Range
Red
Orange
Yellow
< 15
≥ 15 and < 20
≥ 20 and < 30
> 30 Green
Note:
For more information on KB Basecaller and Quality Values, see the Applied
Biosystems DNA Sequencing Analysis Software v5.1 User Guide, PN 4346366.
The column is empty (white) if:
• Analysis was not performed
• Analysis failed
• ABI Basecaller was used for analysis. This basecaller does not assign
Quality Values.
Results Group and Analysis Protocol Columns
The Results Group and the Analysis Protocol (Analysis Method in the GeneMapper
™ software) can be edited and the changes used for reextraction.
Note:
Select an entire column in the Reextraction window by clicking on the column header. For example, clicking on the Extract column header selects all samples. Clicking the Uncheck or Check buttons at the bottom of the window, enables or disables the checkboxes for each sample. Additionally, the fill-down command (Ctrl+D) works the same here as in the Plate Editor for easier information input.
Sorting The Samples
The samples can be sorted according to any of the column properties by holding down the shift key while clicking on the column header. Shift-clicking again sorts them in the reverse order. This is most useful for sorting by capillary number, by well position, by results, by quality, and by the Extract column. For example, it is often useful to bring all of the samples that failed analysis or extraction to the top of the column where they can be examined without having to scroll down to each sample individually.
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Reextracting Selected Samples
1.
Expand the Results column cells for any yellow or red results, to see a description of the warning or failure.
2.
If desired, select a new Results Group, or edit the current one. This allows you to turn off autoanalysis, change the samples and folder naming options, the location where they are placed, the owner of the Results Group, etc.
3.
If desired, change the Analysis Protocol to experiment with different ways of analyzing the sample, using a different basecaller for example.
4.
Check the check box in the Extract column for the samples you wish to extract again.
5.
Click Extract.
IMPORTANT!
Reextraction creates an entirely new sample file and does not replace the previously saved sample file. The presence of a previous sample file has no effect on the creation of a new sample file. If the same naming options that are used for reextraction are identical to those used previously, a number is appended to the filename. For example, if the first sample is,
“sample 01.ab1” then the second sample would be, “sample 01 (1).ab1.”
Viewing the Results of Autoextraction
Reextracting Selected Samples
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Viewing the Results of Autoextraction
Notes
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Performing Maintenance
Workflow
Preparing the
Instrument
Performing
Spatial Calibration
Performing
Spectral Calibration for Sequencing and
Fragment Analysis
Setting Up the Software for
DNA Sequencing
Setting Up the Software for
Fragment Analysis
Running the Instrument
Performing
Maintenance
Audit Trails and Access Control
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Clean the pump block and lower polymer block
Flush and fill the water trap
Perform a short-term shutdown
Store a capillary array
Archive data
Defragment the computer hard drives
7
Delete records from the databse
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Performing Maintenance Tasks
Performing Maintenance Tasks
Overview
This section lists common tasks required to maintain your Applied Biosystems
3730/3730xl DNA Analyzer in good working condition. The tasks are divided into tables based on how often you should perform each task.
Wear appropriate protection, including gloves, laboratory goggles, and coat whenever you work with the fluids used on this instrument, or parts that may come into contact with these fluids.
Daily Tasks
Perform these tasks at least once per day.
Maintenance Task
Ensure adequate levels of buffer and water in reservoirs.
Ensure the plate assemblies are properly assembled.
IMPORTANT!
The holes in the plate retainer must align with the holes in the septa, or the capillary tips will be damaged.
Ensure the plate assemblies are positioned on the plate deck properly.
Plates should sit snugly on the deck.
IMPORTANT!
Never use warped plates.
Check the level of buffer in the buffer jar and ensure that the overflow hole is not occluded and, that the overflow hole is facing toward the front of the instrument.
Replace the water and 1X run buffer in the reservoirs on the instrument and, make sure that the outside of the assemblies are dry.
Check for bubbles in the pump block, lower polymer block, interconnect tube, polymer supply tube, and channels.
Remove all bubbles with the Bubble Remove wizard.
Check the loading-end header to ensure the capillary tips are not crushed or damaged.
Check the level of polymer in the bottle to ensure sufficient volume for runs.
Check the pump block and the lower polymer block to ensure they fit securely on the instrument.
Clean the instrument surfaces.
Check for leaks around the array knob, interconnecting tube nuts, and check valve.
Frequency
Before each run
Before each run
Before each run
Before each run
Every 48 hours
Daily or before each run
Daily or before each run
Daily or before each run
Daily
Daily
Daily
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Performing Maintenance Tasks
Weekly Tasks
Perform these tasks at least once per week.
Maintenance Task
Replace the polymer using the Change Polymer Wizard.
Check the storage conditions of the used arrays.
Monthly Tasks
Perform these tasks at least once per month.
Maintenance Task
Run the Water Wash Wizard.
Flush the array port during this wizard, whether or not bubbles are present in the array port.
Flush the water trap (see
).
Frequency
Weekly or as needed
Weekly
Frequency
Monthly or as needed
Monthly or as needed
As-Needed Tasks
Perform these tasks as needed.
Maintenance Task
Clean the drip tray.
Change the array.
Remove any dried polymer from the capillary tips. Use a lint-free wipe moistened with deionized water.
Frequency
As needed
As needed
As needed
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Chapter 7 Performing Maintenance
Performing Maintenance Tasks
General
Instrument
Cleaning
To clean the instrument:
1.
Ensure the oven door, the instrument door, and the stacker are closed.
2.
Press the Tray button on the front of the instrument to move the autosampler to the forward position.
3.
Wipe off any liquid on or around the autosampler using a lint-free tissue.
4.
Clean out the drip tray with deionized water and lint-free tissue.
5.
Clean off any polymer build-up (crystals) on the instrument including the capillary tips with deionized water and lint-free tissue.
IMPORTANT!
its components.
Never use organic solvents to clean the instrument or any of
Notes
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Guidelines for Pump Block and Lower Polymer Block Cleaning
Cleaning Exterior Surfaces
Guidelines for Pump Block and Lower Polymer Block
Cleaning
Cleaning Exterior Surfaces
• Do not expose the polymer blocks to organic solvents.
• Do not use sharp or pointed instruments to remove dried polymer from the polymer blocks.
• Do not use water > 50 °C to clean the polymer blocks.
CHEMICAL HAZARD. POP-7
polymer may cause eye, skin, and respiratory tract irritation. Please read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. Use for research and development purposes only.
When to Clean the Pump Block and the
Lower Polymer Block
• Clean the exterior every 7 days, when polymer is replenished.
• Clean the Polymer Delivery Pump (PDP) chamber, channels, and tubing once per month.
Cleaning the PDP Chamber, Channels and
Tubing
1.
Run Water Wash Wizard.
2.
Inspect the channels of the Pump and Lower blocks for any possible contaminants. Repeat
Water Wash Wizard until contaminants are removed.
Polymer Block Cleaning
Su M T W Th F S
7 days
7
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Chapter 7 Performing Maintenance
Wizards
Wizards
Overview
The five wizards in the Data Collection Software v2.0 guide you through several maintenance procedures.
General Use Guidelines
The following table lists the wizards and when to use them.
Wizard
Install Array
Change Polymer
When to Use
• To install a capillary array:
– On a new instrument
– To reactivate an instrument that has been shut down
• To replace an installed capillary array with another capillary array
• To install an array without the wizard when the Data Collection software is reinstalled or upgraded
• To replenish the polymer supply
• To replace the polymer in the PDP with polymer of the same or different lot
• To enter polymer information when Data Collection software is installed or upgraded
Bubble Remove • To remove bubbles in the PDP chamber, channels, and tubing
Water Wash
Instrument
Shutdown
• To wash the PDP chamber, lower polymer block*, channels, and tubing with water:
– As part of a monthly maintenance protocol
– To remove any suspected contaminants in the PDP
– To remove persistent bubbles (followed by the Bubble Remove Wizard, if needed)
– To replace old polymer in the PDP
* The lower polymer block should not be removed; clean on the instrument using this wizard.
• To prepare the instrument for a period of disuse of greater than one week
• To remove the array without the wizard
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Wizards
General Use Guidelines
Using Wizards with the
Instrument Door
Open
IMPORTANT!
Polymer Delivery Pump (PDP) operations with the door open are permitted only in wizards. If the door is opened at any other time during instrument operation, most functions, including PDP operations, are paused.
All wizards include instructions for manual operations and buttons that launch specific, automated operations of the Polymer Delivery Pump (PDP). You can use the
3730/3730xl version 2.0 to perform most automated operations of the wizards with the main instrument door open. With the door open, you can easily view the operations of the PDP, making possible close monitoring of bubble clearing and fluid changing. The wizards may also be performed more quickly, since the instrument door need not be closed (triggering a wait period for the green status light to illuminate) and opened repeatedly when the automated procedures are performed. Guidelines for using the capability to run automated wizard procedures with the door open are given in the guidelines table in the next section.
Guidelines for
Wizard Use
Depending on
Instrument Door
State
Follow the suggestions below to use the wizards effectively when the door is open or closed.
IMPORTANT!
Whenever the door is closed (whether or not a wizard is open, or an automated procedure is in progress), the autosampler moves while determining position (initialization). Always wait for the autosampler to stop moving (finish initialization), and the green status light to illuminate before initiating any automated procedures. If you accidentally start an automated procedure while the autosampler is in motion, you may encounter irregularities in the instrument behavior. An error may be displayed in the Data Collection event window.
However, you should be able to complete the Wizard. Restart the instrument and the Data Collection software.
If You...
With the
Instrument Door ...
Then ...
IMPORTANT!
Do not open or close the instrument door while an automated procedure is in progress. Leave the door in the starting state (whether open or closed) until the automated procedure is complete.
Begin an automated procedure
Open The procedure continues when the door is closed, and after the autosampler moves to initialize. If you open the door again, the procedure pauses until the door is closed.
Begin an automated procedure
Closed The procedure pauses if the door is opened. Close the door again to resume the procedure.
Click Fill Array
Perform an automated procedure
Open
Closed
The procedure does not start; the door must be closed.
The green status light remains on (not flashing)
Perform an automated operation
Open
The yellow status light flashes.
Note:
Regardless of whether or not automated procedures are in progress during wizard use:
• If the instrument door is closed, then the green status light remains on (not flashing)
• If the instrument door is open, then the yellow status light flashes
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Wizards
Specific Use Guidelines
If ...
The polymer has been in the pump longer than 1 week
Bubbles move but are not completely cleared by the
Bubble Remove
Wizard
You want to clear
persistent bubbles
Then ...
Explanation
Use the Water Wash Wizard (instead of Change Polymer Wizard) to replace the polymer.
Use the Bubble Remove Wizard
repeatedly until the bubbles are gone.
Using the Water Wash Wizard ensures that the system is well cleaned before fresh polymer is introduced.
Urea decomposition can cause an increase in electrophoresis current in polymer that has been at room temperature for more than 1 week.
When clearing bubbles with repeated use of the wizard, note whether or not the target bubbles have moved during performance of the Wizard. Any bubbles that move but are not entirely cleared by running the wizard are likely to be cleared with a repeat of the Bubble Remove Wizard.
Try one or both of the following:
• Run the Water Wash Wizard followed, if necessary, by the Bubble Remove Wizard. (The Water
Wash Wizard includes refilling the pump with polymer.)
• Remove the polymer bottle and run the Bubble Remove Wizard (a large amount of air will be drawn into the pump chamber and other parts of the system). Reinstall the polymer bottle and repeat the Bubble Remove Wizard to remove all bubbles.
Note:
If the pump sits idle for a time, bubbles that previously did not move are often cleared by running the
Bubble Remove Wizard.
The Water Wash Wizard may help to remove bubbles.
Many, or large bubbles are present in the pump chamber
No bubbles are present in the array port during the monthly water wash procedure
You want to install a capillary array on an instrument without an array
You remove or install a capillary array
You should still perform the Flush
Array Port procedure using the Water
Wash Wizard as part of monthly maintenance, even if no bubbles are present.
Perform the Fill Array procedure at the end of the Install Array Wizard.
Carefully follow the instructions in the appropriate wizard (Install Array or
Instrument Shutdown wizards).
Ensure that the instrument configuration and the database information agree.
The information for that array cannot be entered again on the instrument.
You select Discard during installation of an array using the
Install Array Wizard
You plan to leave the instrument unused for more than 1 week
Use the Instrument Shutdown
Wizard.
Occasional flushing of the array port keeps this space filled with fresh solution.
Filling the array helps to ensure complete changeover to polymer in this situation where the PDP has been extensively washed with water.
A mismatch between the array configuration/identification and the database information may cause incorrect analysis parameters and result in reduced basecalling accuracy.
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Wizards
Specific Use Guidelines
If ...
You are using the
Install Array Wizard to reactivate the instrument
You cancel a wizard during an automated procedure
You want to move the pump block on the mounting pins
You want to clean the array port knob, plug, or opening
You want to clean the PDP with the
Water Wash wizard
Then ...
First turn on the instrument power to activate the wizard menu in Data
Collection.
The piston motion in progress will finish before the wizard is terminated.
Grasp the body of the pump, not the water seal fixtures.
Carefully clean the threads of these parts with moistened lab wipes.
Use only deionized water at
≤ 40 °C
Explanation
The instrument must be powered for the wizards to be available through Data Collection. If the instrument is turned off, the wizard names in the pull-down menu are grayed out.
The piston cannot stop immediately. During the period between cancellation and termination while the piston is in motion, a “Please wait...” dialog box displays.
Excessive force can damage the water seal fixtures.
Dried polymer deposits on the threads may cause poor sealing when the parts are rejoined.
Hot water may damage the PDP seals and joints. Do not use any solutions or fluids in the instrument other than water and polymer.
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Chapter 7 Performing Maintenance
Wizard Flowcharts
Wizard Flowcharts
The five wizards are depicted below as flowcharts.
Each flowchart shows decision branches and alternative pathways.
Install Array Wizard
If an array is not installed
Start the Install Array Wizard
Install an array for instrument reactivation
Install an array for new PDP
If an array is installed
Start the Install Array Wizard
Store an array Discard an array
Verify the instrument configuration
Enter array information
Install water bottle
Fill the array with polymer
Remove and store the array
Remove and discard the array
Enter array information
Install the array
Wash the pump, channels, and tubing with water
Enter polymer information
Install polymer
Finish
Flush
Are bubbles present?
NO
YES
Remove bubbles
Are bubbles present in the array port?
NO
YES
Loosen array knob
Flush array port
Tighten array knob
Does the array need to be filled with polymer?
YES
NO
Finish
Fill array
Install the array
Prime the pump with fresh polymer
Finish
Are bubbles present?
NO
YES
Remove bubbles
Are bubbles present in the array port?
NO
YES
Loosen array knob
Flush array port
Tighten array knob
Does the array need to be filled with polymer?
YES
NO
Finish
Fill array
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Change Polymer Wizard
Start the Change Polymer Wizard
Install polymer from a different lot
Install polymer from the same lot
Enter polymer information
Replace polymer
Prime the pump with fresh polymer
Are bubbles present?
NO
YES
Remove bubbles
Are bubbles present in the array port?
NO
YES
Loosen array knob
Flush array port
Tighten array knob
Does the array need to be filled with polymer?
YES
NO
Finish
Fill array
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Wizard Flowcharts
Change Polymer Wizard
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Chapter 7 Performing Maintenance
Wizard Flowcharts
Bubble Remove Wizard
Start the Bubble
Remove Wizard
Remove bubbles
Are bubbles present?
NO
YES
Remove bubbles
Are bubbles present in the array port?
YES
NO
Loosen array knob
Flush array port
Tighten array knob
Does the array need to be filled with polymer?
YES
NO
Finish
Fill array
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Water Wash Wizard
Start the
Water Wash Wizard
Install the water bottle
Wash the pump, channels, and tubing with water
Are you installing polymer from the same lot?
YES
NO
Enter polymer information
Replace polymer
Flush with fresh polymer
Are bubbles present?
NO
YES
Remove bubbles
Are bubbles present in the array port?
YES
NO
Loosen array knob
Flush array port
Tighten array knob
Does the array need to be filled with polymer?
YES
NO
Finish
Fill array
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Wizard Flowcharts
Water Wash Wizard
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Chapter 7 Performing Maintenance
Wizard Flowcharts
Instrument Shutdown Wizard
Store the array
Start the Instrument Shutdown Wizard
Discard the array
Shut down without the wizard
Fill the array with polymer
Flush the pump, channels, and tubing with water
Remove and store the array
Clean the buffer jar
Clean the reservoirs
Flush the pump, channels, and tubing with water
Remove and discard the array
Clean the buffer jar
Clean the reservoirs
Finish
Finish
Finish
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Flushing and Filling the Water Trap
Overview
The PDP water trap should be flushed with either distilled or deionized water at least once per month to wash out any diluted polymer, and to clear bubbles.
Leave the trap filled with
e
ither distilled or deionized water.
To flush the water seal trap:
1.
Fill the supplied 20 mL, all-plastic Luer lock syringe (PN 4324463) with distilled or deionized water. Expel any bubbles from the syringe.
Note:
Do not use a syringe smaller than 20 mL.
Doing so may generate excessive pressure within the trap.
2.
Attach the syringe to the forward-facing Luer fitting at the top of the pump block. Hold the fitting with one hand while threading the syringe onto the fitting with the other hand.
3.
Open the Luer fitting by grasping the body of the fitting and turning it and the attached syringe approximately one-half turn counterclockwise.
4.
Open the exit fitting at the top left side of the pump block by turning it approximately one-half turn counterclockwise.
5.
Hold an empty tube or beaker under the exit fitting to receive approximately 5 mL of waste.
Flush the trap by pushing steadily on the syringe plunger.
IMPORTANT!
DO NOT USE EXCESSIVE
FORCE when you push the syringe plunger as this may damage the trap seals. Take approximately 30 seconds to flush 5 mL of either distilled or deionized water through the trap.
Flushing and Filling the Water Trap
Overview
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Chapter 7 Performing Maintenance
Storing a Capillary Array
Note:
Because the water trap volume is approximately 325
µL, a relatively small volume of water is adequate for complete flushing.
However, a larger volume only improves flushing, as long as force and flow rate are kept within the limits given above
6.
Close the fittings in this order by turning each clockwise until the fittings seal against the block:
a.
b.
Luer fitting, first
Exit fitting, second
IMPORTANT!
Do not over-tighten the fittings.
Very little pressure develops within the trap during pump operation, so the fittings require only enough tightening to prevent water leaks.
Excessive tightening can damage the fittings.
7.
Remove the syringe from the Luer fitting. Hold the fitting with one hand while turning the syringe counterclockwise with the other hand.
Storing a Capillary Array
IMPORTANT!
Wear appropriate protection including gloves, laboratory goggles and coat whenever you work with the fluids used on this instrument or parts that may have come in contact with these fluids.
1.
Remove the capillary array from the instrument using the Install Array wizard.
If You ...
Then ...
Follow the Install Array
Wizard instructions
Remove the array without using the Array Wizard instructions
The capillaries are filled with fresh polymer and some of the steps described below will be complete when the array is removed.
The capillaries should first be filled with fresh polymer.
To maintain serviceability during storage, keep both ends of the capillary array immersed in 1X run buffer. Failure to do so may result in array damage.
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Storing a Capillary Array
Overview
2.
Put 80 mL of 1X run buffer in the capillary array header shipping cover.
3.
Lower the capillary tips of the array header into the shipping cover and lock the header onto the cover. The tips of the capillaries should be immersed in buffer.
4.
Clip the detection cell window cover onto the detection cell.
5.
Attach the detection cell with its cover to the storage post on the array frame.
6.
If the array knob and double-tapered ferrule are on the array tip:
a.
Remove them and rinse them with deionized water.
b.
Dry the parts with a lab wipe.
c.
If they are not to be used immediately, store them in a safe place.
7.
Clean the array tip carefully with a lab wipe moistened with deionized water.
8.
Attach the array tip shipping vial filled with 1X run buffer to the array tip. Loosen the vial cap slightly, insert the tip and then tighten the cap.
9.
Clip the vial with the array tip onto the array frame.
10.
Store the capillary array upright in a safe area.
IMPORTANT!
Check the 1X run buffer levels in the shipping cover and vial at least once a week; replenish the buffer as necessary to keep the both ends of the capillaries immersed in buffer.
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Chapter 7 Performing Maintenance
Performing a Short-Term Shutdown
Performing a Short-Term Shutdown
Perform the short-term shutdown procedure if you will use the instrument again in 7 days or less.
Su M T W Th F S
Materials Required
• 1X Run Buffer
• POP-7 polymer
• Deionized water
• Lab wipes
• Gloves
Performing a Short-Term Shutdown
1.
Close the instrument door.
≤ 1 week
Instrument door
2.
Press the tray button to bring the autosampler to the forward position.
3.
Wait for the:
• Autosampler to stop moving
• Green status light to illuminate then open the instrument door.
4.
Remove the buffer, water, and waste reservoir assemblies from the instrument.
5.
Disassemble each reservoir assembly and empty the contents of the reservoirs into an aqueous waste container.
Aqueous
Waste
Status lights
Tray button
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6.
Rinse each reservoir using deionized water.
7.
Dry the reservoirs using lint-free wipes.
Performing a Short-Term Shutdown
Overview
DI H
2
O
≤ 50 °C
8.
Fill and assemble the reservoirs.
Buffer Reservoir Assembly
1. Add 80 mL 1
✕ running buffer to the Buffer reservoir.
2. Assemble the reservoir assembly as shown below:
Water and Waste Reservoir Assemblies
1. Add 80 mL high-quality deionized water to each reservoir (Water and Waste).
2. Assemble each reservoir assembly as shown below:
Retainer
Septa
Reservoir cap
Reservoir
Heated plate base
Power cable
GR2210
9.
To prevent damage to the capillary array, inspect each reservoir assembly and verify that the:
• Septa fit snugly and flush on the reservoir
• Rubber gasket around the edge of the reservoir cap is seated
Retainer
Septa
Reservoir cap
Reservoir
Plate base
Rubber gasket not seated correctly
• Plate retainer holes and the septa strip are aligned
Plate retainer holes and septa holes are not aligned
7
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Performing a Short-Term Shutdown
10.
Dry the reservoirs using lint-free wipes.
Lint-free wipe
11.
Connect the Buffer reservoir plate base cable into the heater outlet within the instrument.
IMPORTANT!
After placing the buffer reservoir, make sure the cable is out of the way of the autosampler.
12.
Place the Water and Waste reservoirs into the instrument. All three reservoirs will be in the following order:
a.
b.
Buffer reservoir
Water reservoir
c.
Waste reservoir
13.
Close the instrument door.
14.
Press the tray button.
12a 12b 12c
Buffer reservoir
Buffer position
Instrument door
15.
Press the instrument power button.
Power button
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16.
Shut down the computer:
a.
b.
c.
Select > Shutdown.
In the Shut Down Windows dialog box, select Shut down from the drop-list.
Click .
17.
Press the power button on the monitor.
Performing a Short-Term Shutdown
Overview
19b
19c
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Chapter 7 Performing Maintenance
Working With Drives for Database and Sample Data Storage
Working With Drives for Database and Sample Data Storage
Checking
Available Space on Drives D, E, and F
Before a run or batch of runs, the Data Collection software automatically checks the available space to ensure sufficient space to store the database and sample file data you create.
The Data Collection software sends a warning message to remove data when the drive is getting full and/or clean up the database when the database is getting full (~80% of capacity). An error is generated and displayed in the Instrument Status window in the
Errors pane and in the Event Log window in the Errors pane. Also, the status light in the bottom left-hand corner of the data collection window flashes red.
Full Database
Error
To view the error messages, click GA Instruments > ga3730 >
instrument name > Instrument Status> Event Log.
IMPORTANT!
Runs can not be started until the data is removed from the drive and/or database is cleaned up.
Status light
Cleaning Drives
Ensure that you have sufficient drive space by regularly:
• Archiving data
• Emptying the recyle bin
• Deleting unneeded files
• Defragmenting the drives
Notes
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Hard Disk Status
Checking Available Disk Space on Drive E
Hard Disk Status
Checking Available Disk Space on Drive E
1.
In the Tree pane of the Data Collection Software, click
GA Instruments > Database Manager.
The Database Manager view opens.
Check disk space status here
Checking available drive E space
2.
If there is insufficient space:
a.
Archive the sample files to a CD-RW (see page 176
) or another volume.
b.
Delete the sample file data from the drive E and empty the contents of the
Recycle Bin.
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Archiving Data
Archiving Data
Creating a Data CD
A basic version of Roxio Easy CD Creator™ 5 software was loaded on your Dell™ computer. Use this software to archive data to a CD. The software is also part of the CD set you received with your Dell computer.
To archive data:
1.
Select Start > Programs > Roxio Easy CD
Creator 5 > Applications > Easy CD Creator.
The Untitled - Easy CD Creator dialog box opens.
2.
For help creating a data CD, select Help >
Contents and Index.
3.
In the left tree pane, select Making Data CDs
for Archiving and Sharing > Making a Data
CD.
Use the instructions to create the CD.
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Archiving Data
Creating a Data CD
Instructions for creating a data CD
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Defragmenting the Computer Hard Drives
Defragmenting the Computer Hard Drives
The fragmentation of files decreases the performance of both the data collection software and the computer operating system. As the hard drive becomes fragmented, programs take greater time to access files because they must perform multiple seek operations to access the fragments.
When to Defragment the Computer Hard Drive
Defragment the computer hard drive:
• at least once every month.
• before fragmentation reaches 10%.
Defragmenting the Hard Drive
1.
In the Windows desktop, right-click
My Computer ( ), then select Manage.
2.
In the Tree tab of the Computer Management dialog box, click
Computer Management
(Local) > Disk Fragmenter.
3.
Select the E drive.
4.
Click .
The computer displays the Defragmentation
Complete dialog box upon completion of the defragmentation of the drive.
5.
In the Defragmentation Complete dialog box, click .
6.
In the Computer Management dialog box, click .
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Deleting Records from the Database
Deleting Processed Frame Data
Deleting Records from the Database
Deleting Processed Frame Data
1.
In the Tree pane of the Data Collection Software, click GA Instruments >
Database
Manager.
The Database Manager view opens.
IMPORTANT! The Cleanup Database utility deletes all run data and plate records in the database. Before running the utility, be sure that all runs have been extracted from the database.
2.
Click Cleanup Processed Plates.
The following dialog box opens.
3.
Click .
Note:
There is no need to re-import the spatial and spectral calibrations or the custom run modules.
Note:
It may take several minutes to clean up the database if it is full or contains a lot of data.
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Deleting Records from the Database
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Audit Trails and Access Control
Workflow
Preparing the
Instrument
Performing
Spatial Calibration
Performing
Spectral Calibration for Sequencing and
Fragment Analysis
Setting Up the Software for
DNA Sequencing
Setting Up the Software for
Fragment Analysis
Running the Instrument
Performing
Maintenance
Audit Trails and Access Control
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Enable the access control and audit features
Start the AB Navigator
Audit
Configure the audit map
View the audit history
Access Control
Export/import user settings, applications, group profiles
Set up password policies
Create a new user
Create a new profile
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Audit
Audit
Audit trails maintain a history of data changes made by the user.
Data Changes that Generate
Audit Records in
Data Collection
Software
An audit record is generated when data are changed. The following table lists the three general categories and the events within them that generate an audit record in Data
Collection software.
An audit record is generated in Data
Collection software when you ...
Plate Record
• Create, edit, or import a plate record
Run Module
• Create, edit, or import a run module
Results Group
• Create, edit, or import a results group
Reason For
Change
When a change occurs and auditing is required, the Reasons For Change dialog displays and contains:
• The attribute that was created or changed.
• The old and new values, if applicable, in the top half of the dialog box.
• A Text box to enter the reason for the change.
– When you click OK, changes to the attribute and the audit data are saved.
– When you click Cancel, no changes are saved and you return to the previous window.
Parameter changes
Reason for changes
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Enabling The Access Control and Audit Features
Enabling Access Control (Security)
Enabling The Access Control and Audit Features
Enabling Access Control (Security)
1.
Start the following:
– Data Collection services: Start > Programs >
Applied Biosystems > Data Collection >
Run Data Collection 3730 v2.0.
– Administrator application: Start > Programs
> Applied Biosystems > Administrator.
2.
In the System Authentication dialog box, type
Administrator for the login name and type your password if you have changed it; if not, type
Administrator.
3.
In the left pane tree double-click Access Control
Administration.
4.
Select Applications > FoundationViewerApp.
5.
Select the Challenge checkbox to activate it.
The Login and Password dialog box is now enabled.
6.
Select File > Save.
7.
Exit Access Control Administration
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Enabling The Access Control and Audit Features
Enabling Audit
1.
In the Navigator left pane tree, double-click
Audit Map Configuration.
2.
Select the Enabled checkbox for each audit map to activate it:
• DC Plate Record
• DC Run Module
• DC Results Group
3.
Exit Audit Map Configuration.
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Starting AB Navigator
AB Navigator is the access point for these applications:
• Audit Map Configuration
• Audit Map History Viewer
• Access Control Administration
IMPORTANT!
You must start Data Collection services in order for AB Navigator to function properly.
1.
Start the Data Collection Services, then start the
Administrator application:
a.
b.
Data Collection services: Start > Programs
> Applied Biosystems > Data Collection >
Run Data Collection 3730 v2.0.
Administrator application: Start >
Programs > Applied Biosystems >
Administrator.
The System Authentication dialog box displays.
2.
Enter login name and password and click OK.
Default login name: “Administrator”
Default password: “Administrator”
Note:
To change your password, see
Starting AB Navigator
Enabling Audit
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Audit Map Configuration
3.
In the left pane tree, click Administration to expand the options.
Audit Map Configuration
The Audit Map Configuration Tool is used to manage
Audit Maps. Audit Maps are used to control how auditing is done for a given functional area.
Some features of the Audit Map Configuration Tool:
• You can set the audit states of an audit map to
On, Off, or Silent.
• There is no SAVE command. All changes to audit maps are saved automatically.
Starting the Audit Map Configuration Tool
1.
Double-click the Audit Map Configuration icon in the left pane tree.
The System Authentication dialog box displays.
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2.
In the System Authentication dialog box, type
Administrator for the login name and type your password if you have changed it; if not, type
Administrator.
3.
Click OK.
The Audit Map Configuration window displays.
Audit Map Configuration
Audit Map Configuration Functions
Audit Map Configuration Functions
Audit Map Configuration Functions:
If you want to ...
Enable or disable all the attributes in an audit map
Then ...
Select or deselect a cell in the Enabled column in the Audit Map Objects pane.
Sort a row Click on a column header.
Note:
Disabled Audit Maps (Enabled column) display their attribute list in italics.
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Audit Map
Objects
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Audit Map Configuration
Commands
The following table lists the commands you can perform in the Audit Map Configuration
Tool.
File
Toolbar
Menu
Command Function
Auditing
Go To Displays a list of applications that are currently running; select an application to go to that application
Visual Print
Visual Print
Preview
Displays Print Dialog
Displays Print Preview
Exit Application Exits the Audit Map Configuration application
Exits the AB Navigator application Exit AB
Navigator
On Select auditing to be turned on for the Audit Map
Configuration.
When a change is made to an Audit Map’s enabled state or when a change is made to the state of an attribute, auditing occurs, and A Reason For Change (RFC) dialog displays.
When RFC Dialog
Displays and You...
Click OK
Click Cancel
Then ...
The map or attribute state changes and an Audit
Record is created.
The map or attribute state does not change.
Silent When a change is made to an Audit Map’s enabled state or when a change is made to the state of an attribute, auditing occurs. Although the RFC Dialog does not display, a ‘silent’ Audit Record is created.
Note:
Changes to the auditing state from this toolbar menu item (Auditing) applies only to the Audit Map Configuration tool. To change the auditing states of Data Collection software functional areas, see the section below.
Applied Biosystems 3730/3730xl DNA Anaylzer User
Audit Map Configuration
Attribute States
Attribute States
When you click an Audit Map Object, the Attributes Pane displays.
Change
Description
This function controls the Reason for Change dialog box. When it is on, any changes to the enabled Audit Map Object forces the user to type a reason for the change.
To disable this feature for an enabled object—The DC Results Group in the graphic above—change the state to Off.
Parameter
Change
This function records old and new values that are displayed in the upper half of the
Reason for Change dialog box (see “Reason For Change” on page 182 ).
On, Off, and Silent
The following table describes the On, Off, and Silent states for audit map attributes,
Change Description and Parameter.
State
On
Off
Silent
Audit Map Attributes
Change Description
Reason for change required
Reason for Change dialog box does not display
Reason for Change dialog box does not display
Parameter
Records old and new values
Does not record old or new value changes
Records old and new values
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Audit History Viewer
Audit History Viewer
The Audit History Viewer is used to view historical audit data. This tool is used as a read-only viewer for audit records. The tool provides data filtering so that audit records can be viewed in different formats.
Audit records that you can view with the Audit
History Viewer are:
• Date and time the audit record was created.
• The user who triggered the audit event.
• The attribute that was changed.
• The old and the new values.
• The reason for the change.
Note:
The audit records are stored in a permanent data store.
Starting the Audit History Viewer
1.
Double-click the Audit History Viewer icon in the left pane tree.
2.
In the System Authentication dialog box, type
Administrator for the login name and type your password if you have changed it. If not, type
Administrator.
3.
Click OK.
The Audit History Viewer displays.
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Viewing an Audit History
1.
In the Audit Objects pane, expand the objects tree until the object of interest displays.
Audit History Viewer
Viewing an Audit History
2.
Highlight an object and then click (Detail
Panel) to display audit record details.
Note:
Click the column headers to sort the readonly records columns.
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Audit History Viewer
Filter Command
The filter allows you to categorize audit history records.
1.
Click
The Filter Audit Records pane displays.
2.
Enter search criteria in the applicable text boxes.
3.
Click Find Now.
You can filter audit records by:
• Name
• Date (and, before or after a date or between two dates)
• User name
• Matching whole words
• Case sensitivity
Commands
Toolbar
Menu
File
View
Reload
Report
Print Preview
Command
Page Setup
Go To
Visual Print
Visual Print Preview
Exit Application
Exit AB Navigator
Filter
Function
Refreshes the Audit History Viewer with the latest changes
Customize and then print a report of the selected Audit
History Record
Customize and then preview a report of the selected Audit
History Record
Customize the page setup of the Report printout
Displays a list of applications that are currently running; select an application to go to that application
Displays Print Dialog
Displays Print Preview
Exits the Audit Map Configuration application
Exits the AB Navigator application
Displays the filter pane on the top of the frame when selected. It allows the user to specify criteria that limits the amount of audit records in the Audit Record table.
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Access Control Administration
The Access Control Administration tool allows an administrator to manage the creation and deletion of:
• Users
• Profiles
Also, Access Control allows an administrator to restrict or grant users access to features and functions of the software.
An administration user is always associated with the
Administration User Group and cannot be deleted.
And, only one administrator is allowed to modify
Access Control data at one time.
Starting the Access Control Administration Tool
IMPORTANT!
You must start Data Collection services in order for AB Navigator to function properly.
1.
Start the following:
– Data Collection services: Start > Programs >
Applied Biosystems > Data Collection >
Run Data Collection 3730 v2.0.
– Administrator application: Start > Programs
> Applied Biosystems > Administrator.
2.
Double-click the Access Control Administration icon in the left pane tree.
3.
In the System Authentication dialog box, type
Administrator for the login name and type your password if you have changed it. If not, type
Administrator.
Access Control Administration
Starting the Access Control Administration Tool
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Access Control Administration
4.
Click OK to display Access Control
Administration
Access Control Administration Tool Bar
Icon
Save
Command
Saves changes
Reload
Report
Function
Refreshes the Access Control Administration window with the latest changes
Customize and print a report of the selected Access Control Indentifiers
Duplicate
Delete
Find
New User
New Application
Duplicates the selected entity
Deletes the selected entity
Finds a specific identifier
Opens a new user window
Create a new application (not needed for Data Collection software)
New Profile
Expand
Collapse
Opens a new profile window
Expands all tree nodes making all items visible
Collapses all tree nodes making only the root tree items visible
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Exporting User Settings, Applications, and Group Profiles
Access Control Administration Tool Bar
Exporting User Settings, Applications, and Group Profiles
Exporting and Importing the Access Control
Administration settings can be useful if the same users and group profiles will be shared amongst many computers. This procedure can minimize the time needed to re-create the access control settings on the other computers. The Administrator will only need to create the User, Application, and Group profiles settings on one computer, and then import these settings to the other computers.
1.
Select File > Export Database.
2.
Choose a local or network location and type in a file name for the export file, and click Save.
3.
The Export Users dialog box displays after a successful export.
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Importing User Settings, Applications, and Group Profiles
Importing User Settings, Applications, and Group Profiles
Importing the Access Control Administration settings will over-write any existing User, Application, and
Profile settings. The Audit Map Configuration and
Audit History Viewer will not be affected.
1.
On the computer that you want to import the
Access Control Administration database, enter the Access Control Administration.
2.
Select File > Import Database.
3.
Locate the file that was exported, and click Open.
4.
The Import Users dialog box displays after a successful import.
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Password Policies
1.
Customize passwords by accessing Settings >
Password Policies.
Password Policies
Access Control Administration Tool Bar
The following fields are customizable:
• Max Login Attempts:
Sets the maximum number of failed login attempts allowed before the user account is suspended. The User will not be able to log in again until the suspension time period is over, or the Administrator re-sets the user status to
Active.
• Set User State:
– Remain active - Users are never suspended, even though they exceed the maximum login attempts.
– Suspend for x hour(s) - Users are suspended for the set number of hours if they exceed the maximum login attempts. The user can not login to the Data Collection viewer until the end of the suspension, at which time the User's
"Suspended" status reverts to "Active."
• Password:
– Password Lifetime - At the end of the set period, users will be asked to change their password.
– Password Grace Logins - Sets the number of times that users are able to login with their old password before they are forced to change their password.
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Password Policies
• Password Reusability:
– Password Reuse Period - Sets the number of days that users are prevented from re-using their old password(s). For more information see,
“User Password Change” on page 199 .
– Passwords kept per user - Sets the number of the latest passwords chosen by each user that will be remembered.
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Note:
The password history can be cleared by the Administrator by Clicking Clear
Password History for a particular user. If the user forgets his password the Administrator can reset his password at any time without being required to provide the old password.
Applied Biosystems 3730/3730xl DNA Anaylzer User
User Password Change
Users can change their passwords by going to the
Administrator tool.
1.
Select Settings > Change Password.
2.
Users must type in their old password, and then their new password twice in the Change
Password dialog box.
User Password Change
Access Control Administration Tool Bar
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User Password Change
Type Selection
In the left pane tree, Users and Applications are types.
When you select a type, the List of Users pane displays a list of identifiers of the type selected.
IMPORTANT!
Do not remove any applications from the default list in the left pane tree.
Name Selection
When you highlight a name, properties of that name display in the User Properties pane.
Properties Panes
Access control identifiers have an additional drop-list labeled “Control Properties.” This defines the access level an individual is allowed in the Data Collection software.
Three default profiles and their functional access levels:
• Administrator: Complete access to Instrument
Protocols, Instrument Operation, Instrument
Maintenance
• Scientist: Complete access to Instrument
Protocols, Instrument Operation, Instrument
Maintenance
• Technician: Access to Instrument Operation and
Maintenance
The broad functional access levels are further described below:
• Instrument Protocols include: Run Module
Operations, Results Group Operations, Analysis
Protocol Operations, Instrument Protocol
Operations and Reextraction.
• Instrument Operation include: Plate Operations,
Event Log, and Instrument Control Operations.
• Instrument Maintenance: Spatial Calibration
Operations, Manual Instrument Control, Service
Tools, and Wizards.
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Commands
Frequently used commands appear in the application toolbar. Tool tip help text appears when you place the cursor over a button in the toolbar.
• Save: Commits changes in the Admin Tool to data store and is accessible from the menu bar, keyboard shortcut, or toolbar.
• Exit: Invoked by the standard upper-right-corner control or, by the Files/Exit menu selection. If you have updated memory but have not yet committed changes to data store, the application asks, “Information has been modified, Save changes?” The message box provides buttons for
Yes, No, and Cancel.
indentifier. Duplicate is accessible from the menu bar and toolbar.
• Find: locates the name specified in the text field in the navigator tree
• Print: Prints all or some identifiers in various formats selected from the dialog shown below.
Go to File > Report to display the Print
Options dialog box.
\
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Commands
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Creating a New User
Creating a New User
IMPORTANT!
each new user.
You must set a default password for
1.
Click the New User icon .
The New User dialog displays.
2.
Click Next.
The Configure pane displays.
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3.
Complete the information in the window.
4.
Click Set Password.
The Change Password dialog box displays.
5.
Complete the new password and click OK.
6.
Click Finish to complete the creation of a new user.
7.
Click
8.
Click Next.
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Creating a New User
9.
(Optional) The Show EULA and Pre-Expire checkboxes are selected by default.
• If Show EULA checkbox is enabled, the first time the user logs into the 3730/3730xl
Data Collection Software, the End User
License Agreement displays. Uncheck this option if you do not want to force users to view the EULA.
• If Pre-Expire checkbox is enabled, users are forced to personalize their passwords when they log into the 3730/3730xl Data
Collection Software with the default password for the first time. Uncheck this option if you do not want to give the user the ability to change the default password.
10.
Click
User Properties
A user must be assigned to a profile, which allows the administrator to grant or deny a user the right to execute functions defined by applications.
When one user is selected in the left navigator tree, the user profile displays in the User Properties pane.
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Default Profiles
User Groups
Default profiles show the access each user group has.
The default user groups and their default profiles are:
• Administrator: Complete access to Instrument
Protocols, Instrument Operation, Instrument
Maintenance
• Scientist: Complete access to Instrument
Protocols, Instrument Operation, Instrument
Maintenance
• Technician: Access to Instrument Operation and
Maintenance
Inherited Rights
The broad functional access levels are further described below:
• Instrument Protocols include: Run Module
Operations, Results Group Operations, Analysis
Protocol Operations, Instrument Protocol
Operations and Reextraction.
• Instrument Operation include: Plate Operations,
Event Log, and Instrument Control Operations.
• Instrument Maintenance: Spatial Calibration
Operations, Manual Instrument Control, and
Miscellaneous Operations.
Default access rights for
Technicians
Default Profiles
User Groups
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Default Profiles
Overriding Inherited Rights
Selecting the OIR check box, allows the
Administrator to change the default rights inherited from a parent node. The Scientist Group, for example, is allowed Full System Control, because they have the ability to execute any function contained under the
FoundationDataCollection node. In another words, all the sub nodes under FoundationDataCollection is checked because the Executable rights are “inherited” from the FoundationDataCollection node.
To override the inherited rights of a User Group:
1.
Deselect the OIR check box next to the function you want to change.
2.
Select the Execute check box to allow access to the feature or deselect the Execute check box to deny access to the feature.
To override the inherited rights of a group:
1.
Deselect the OIR check box next to the function you want to deny. In the example to the right, the
Scientist group is denied access to Instrument
Operation.
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Creating a New Profile
1.
Click the New Profile icon
The New Profile dialog displays.
2.
Click Next.
The Configure pane displays.
3.
Complete:
a.
b.
Profile properties.
Select OIR and/or Execute.
Execute: Select this to give access to the function to any user assigned to this Profile.
OIR: Select this to override inherited rights.
Any lower level in the hierarchy inherits the access rights of the node above it.
To override inherited defaults, check the
OIR check box. This allow the administrator to grant or deny a group’s ability to execute a specific function on a lower level of the hierarchy tree.
4.
Click Next.
The Summary pane displays the properties and associations of the new profile name.
5.
Click Finish to complete the creation of a new
User Profile Name.
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Creating a New Profile
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Parts List
A
Description
3730 36-cm capillary array
3730 50-cm capillary array
3730xl 36-cm capillary array
3730xl 50-cm capillary array
3700/3730 BigDye Terminator v3.1 Sequencing Std
3700/3730 BigDye Terminator v1.1 Sequencing Std
Matrix Standard Set DS-33
HiDi formamide - 25 mL
POP-7 (5 bottles of 25ml)
Buffer (10X) with EDTA - 500 mL
Buffer (10X) with EDTA - 4L
96-well Sample Plates w/barcode
96-well Sample Plates, no bar code
96-well plate septa
96-well Plate Base (septa sealed)
96-well Plate Base (heat sealed)
96-well Plate Retainer (Septa sealed)
96-well and 384-well Plate Retainer (heat sealed)
384-well Sample plates with barcode
384-well Plate septa
384-well Plate Base (Septa-Sealed)
384-well Plate Base (Heat-Sealed)
384-well Plate retainer (Septa-Sealed)
Heat Seal film - 3-mil
Part No
4331247
4331250
4331244
4331246
4336943
4336799
4318254
4311320
4335615
4335613
4318976
4306737
N801-0560
4315933
4334873
4334875
4334869
4334865
4309849
4315934
4334874
4334877
4334868
4337570
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Appendix A
Notes
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G5, G5-RCT, Any4Dye, and Any5Dye Dye Sets
B
Dye Sets G5 and G5-RCT For Fragment Analysis
Overview
Even small levels of crosstalk could be a concern for users of the 3730/3730xl instruments who perform fragment analysis as well as for applications with a high dynamic range. In fragment analysis applications that have few sample peaks and varying peak intensities, a crosstalk peak may appear as a real sample peak and be incorrectly identified as an allele. Crosstalk is not a concern with sequencing applications as there is a constant stream of peaks electrophoresing past the detector.
Dye Set G5-RCT
To reduce crosstalk for fragment analysis applications, a new dye set has been created for Data Collection Software v2.0, called dye set G5-RCT (reduced crosstalk). G5-RCT uses the same chemistry as dye set G5 (6-FAM
™
, VIC
®
NED
™
, PET
®
, LIZ
®
dyes). This dye set reduces signal, but reduces potential crosstalk to a greater degree, so the reduction in signal-to-noise ratio is less pronounced than the reduction in signal overall.
Higher concentration peaks can be used without going offscale – this results in a higher dynamic range for the G5-RCT dye set.
Recommendations for Using GS or G5-RCT
Dye set G5-RCT may be particularly helpful for users performing fragment analysis with a 96 capillary array, as well as users interested in applications with a high dynamic range (large peaks much higher than small peaks). Most other users will prefer the G5 dye set.
Applied Biosystems supports:
• Fragment analysis on the 96-capillary array using G5-RCT only
• G5 and G5-RCT on the 48-capillary array.
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Appendix B
Dye Sets G5 and G5-RCT For Fragment Analysis
Please see the chart that follows for more information about the advantages and issues to consider for each dye set.
Dye Set
G5-RCT
G5
Advantages
Advantages:
• Potential crosstalk is reduced, which can improve allele calling accuracy for 96 capillary array or high dynamic range applications
• Higher dynamic range – higher concentration peaks can be used without peaks going off scale
Issues:
• Signals are reduced compared to signals generated with G5 dye set
• It is essential to run a spectral calibration each time the capillary array is replaced or moved in the detection cell heater
Advantages:
• Higher sample signals compared to the G5-RCT dye set, as more light is collected from the CCD
• While we recommend spectral recalibration when the capillary array is replaced or moved in the detection cell heater, spectral recalibration is
needed less often with G5 than with G5-RCT
• Optimized for the highest signal-to-noise ratio
Issues:
• You may observe more crosstalk with this dye set compared to the G5-
RCT dye set, particularly on the 96 capillary array or in high dynamic range applications
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Creating a Spectral Calibration for the Any4Dye or Any5Dye Dye Sets
Creating a Spectral Calibration for the Any4Dye or Any5Dye
Dye Sets
The steps to creating and running a customized 4- or
5- DyeSet is similar to running a supported dye set.
The following example highlights the use of
Any4Dye dye set; it works the same for Any5Dye dye set.
1.
Create a spectral protocol for the 4-Dye dye set, specifying the appropriate protocol parameters.
2.
Click OK to save the spectral protocol.
Note:
Customize the Spectral parameters as needed. For more information see,
.
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Appendix B
Creating a Spectral Calibration for the Any4Dye or Any5Dye Dye Sets
3.
Click New in the plate manager to display the
New Plate Dialog box.
4.
Create a spectral plate for the Any4Dye dye set by completing the New Plate Dialog box.
5.
In the Plate Editor, select the Instrument Protocol that you just created in the previous steps and click OK to save the plate.
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Creating a Spectral Calibration for the Any4Dye or Any5Dye Dye Sets
6.
From the Run Scheduler, add this spectral plate to the Input Stack and run it.
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Appendix B
Creating a Spectral Calibration for the Any4Dye or Any5Dye Dye Sets
7.
Confirm that spectral matrices for all capillaries pass. Override individual capillaries and rename calibration as needed.
Notes
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Regular Runs Using Any4Dye or Any5Dye Dye Sets
Regular Runs Using Any4Dye or Any5Dye Dye Sets
The following example highlights the use of
Any4Dye dye set. This process works the same for
Any5Dye set.
1.
In the Protocol Editor, create a regular instrument run protocol for the Any4Dye dye set, and choose the appropriate default run module template. (You can create a customized run module in the module editor if desired).
2.
In the Plate Manager, create a regular plate, selecting the Any4Dye instrument protocol you created in step 1.
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Appendix B
Regular Runs Using Any4Dye or Any5Dye Dye Sets
3.
In the Plate Editor, select the Instrument Protocol that you just created in step 1, and click OK to save the plate.
Notes
218
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Regular Runs Using Any4Dye or Any5Dye Dye Sets
4.
From the Run Scheduler, add this plate into the Input Stack and run it.
Notes
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
219
Appendix B
Regular Runs Using Any4Dye or Any5Dye Dye Sets
Notes
220
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Index
Symbols
.fsa files generated from GeneMapper-Generic
Numerics
3730/3730xl Data Collection starting
A
access control administration
starting administration tool
analysis protocol creating
creating for autoanalysis
deleting
editing
exporting
importing
anode buffer jar, filling
Any4Dye creating a spectral calibration for
how to use
Any5Dye creating a spectral calibration for
how to use
Applied Biosystems customer feedback on documentation
Technical Communications
archiving, data
array port, illustration of
Array View, viewing data in
assumptions, for using this guide
audit history filter command
viewing
audit map configuration functions
configuration tool commands
managing audit maps
Audit Map Configuration tool starting
auditing reason for change
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Australian standards
auto mode
features
scheduling runs with
available space, checking hard drives
B
bar code scanner safety
biological hazard safety
bold text, when to use
buffer fill-line
buffer jar, illustration of
buffer reservoir assembly
filling
buffer valve pin, illustration of
C
Canadian standards
capillary array illustration of
installing
storing
capillary array knob, illustration of
capillary array tip, illustration of
Cautions, description
check valve diagram of
chemical safety
chemical waste safety
computer checking hard drive space
start up and log on
computer workstation safety
conventions bold text
IMPORTANT!
in this guide
italic text
menu commands
notes
user attention words
221
Index
customer feedback, on Applied Biosystems documents
customizing run modules
D
Dangers, description
data archiving
viewing in array view
Data Collection software starting
database deleting records from
full error
default profiles, software access
deleting records, from hard drives
description
documentation feedback
double-tapered ferrule, illustration of
dye set G5 issues
recommendations for use
dye set G5-RCT issues
recommendations for use
dyeset/primer files list of
E
electrical safety
electrical symbols, on instruments
electrode, illustration of
electromagnetic compatibility standards. See EMC
standards
EMC standards
Australian
Canadian
European
European standards
F
file naming acceptable characters
invalid characters
fill down special
filling, water trap
flushing, water trap
fragment analysis creating required settings for
222
fragment analysis spectral calibration passing, example of
G
GeneMapper
for autoanalysis
plate record
GeneMapper-Generic, .fsa files from
H
hard disk, status of
hard drives checking available space
defragmenting
deleting records from
hazards biological
chemical
physical
solvents
heat-sealed plates
I
Important, description
instrument illustrated parts of
operation, manual vs auto mode
startup
instrument protocol creating for fragment analysis
creating for sequencing
importing
interconnect tube
italic text, when to use
L
labels, safety on instruments
laser safety bar code scanner
laser classification
requirements
launching a run
lower polymer block, illustration of
Luer fitting, illustration of
M
magnifying spatial profiles
spectral profiles
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Index
manual mode scheduling runs using
versus auto mode
menu commands, conventions for describing
mounting pin illustration of
MSDSs description
obtaining
N
new user creating
notes, description
O
O-ring, illustration of
overflow hole, illustration of
P
password
pausing a run
PDP motor cover, illustration of
physical hazard safety
piston, illustration of
plate record creating for sequencing analysis
creating GeneMapper
for fragment analysis
for sequencing analysis
GeneMapper elements of
when to create
plates assembling
components
heat-sealed
septa-sealed
polymer adding
replacing
Polymer Delivery Pump (PDP), illustration of
polymer supply bottle cap with hole, illustration of
polymer supply bottle, illustration of
polymer supply tube illustration of
profile default
passing spatial, examples of
spatial calibration, evaluating
pump block, illustration of
pump chamber, illustration of
R
records, deleting from database
reextraction panel effects of changes made in
reservoirs filling
placing into instrument
results group creating for autoanalysis
creating for sequencing
exporting
importing
run spectral, using Any4Dye with
starting, stopping, skipping, pausing
stopped before autoextraction is complete
using Any4Dye with
using Any5Dye with
run buffer preparing
run history view viewing data in
run modules customizing
editable parameters
selecting for sequencing
S
safety bar code scanner
before operating the instrument
biological hazard
chemical
chemical waste
computer workstation
electrical
instrument
laser
moving and lifting
physical hazard
solvents
safety alert words
safety alerts
Caution
Danger
Important
Warning
safety information for system operators
sources of
safety labels, on instruments
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
223
Index
safety standards
Canadian
European
U.S.
safety symbols, on instruments
sample file name, creating
sample run adding
adding for fragment analysis
septa-sealed plates
sequencing plate editor
run modules
sequencing spectral calibrations passing, examples of
service console, using
settings required for automated fragment analysis
required for automated sequencing analysis
shutdown, performing short-term
software, Data Collection
solvents, safety
spatial calibration evaluating profile
failed
performing
troubleshooting
what it tells you
when to perform
spatial profile magnifying
passing, examples of
spectral calibration evaluating results
performing
spectral viewer
starting a run
troubleshooting
spectral profile magnifying
spectral run using Any4Dye with
spectral viewer 52 standards
EMC
safety
starting
Data Collection software
instrument
run
status, of hard disk
stopping a run
storing, capillary array
224
symbols on instruments electrical
safety
T
Technical Communications contacting
e-mail address
toolbar
training, obtaining information about
troubleshooting flashing yellow light
solid red light
solid yellow light
U
U.S. standards
user attention words, defined
user name
W
Warning, description
waste disposal, guidelines
waste profiles, described
waste reservoir assembly, illustration of
water reservoir assembly filling
illustration of
water seal, illustration of
water trap filling
flushing
illustration of
Applied Biosystems 3730/3730xl DNA Analyzer User Guide
Back Cover
Support
For the latest support information for all locations, go to
http://www.appliedbiosystems.com, then click the link for Support.
At the Support page, you can:
• Search through frequently asked questions (FAQs)
• Submit a question directly to Technical Support
• Order Applied Biosystems user documents, MSDSs, certificates of analysis, and other related documents
• Download PDF documents
• Obtain information about customer training
• Download software updates and patches
In addition, the Support page provides access to worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities.
Headquarters
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Foster City, CA 94404 USA
Phone: +1 650.638.5800
Toll Free (In North America): +1 800.345.5224
Fax: +1 650.638.5884
Worldwide Sales and Support
Applied Biosystems vast distribution and service network, composed of highly trained support and applications personnel, reaches 150 countries on six continents. For sales office locations and technical support, please call our local office or refer to our Web site at www.appliedbiosystems.com.
Applera Corporation is committed to providing the world’s leading technology and information for life scientists.
Applera Corporation consists of the Applied Biosystems and
Celera Genomics businesses.
Printed in the USA, 12/2003
Part Number 4347118 Rev. B

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Key features
- High throughput
- 96-sample capacity
- Automated operation
- Flexible software options
- Compatibility with various reagents