BigDye® Terminator v1.1 Cycle Sequencing Kit

BigDye® Terminator v1.1 Cycle Sequencing Kit
BigDye® Terminator v1.1
Cycle Sequencing Kit
Protocol
DRAFT
August 27, 2002 12:32 pm, 4337036A_v1.1Title.fm
© Copyright 2002, 2010 Applied Biosystems. All rights reserved.
For Research Use Only. Not for use in diagnostic procedures.
Notice To Purchaser: Limited License
A license under the process claims of U.S. patents or their foreign counterpart claims, has an up-front fee component and a runningroyalty component. The purchase price of BigDye ® Terminator v1.1 Cycle Sequencing Kit includes limited, non-transferable
rights under the running-royalty component to use only this amount of the product to practice the DNA sequence and fragment
analysis processes described in said patents when this product is used in conjunction with an Authorized DNA sequence analysis
instrument whose use is covered under the up-front fee component of these patents. No other rights are granted expressly, by
implication, or by estoppel, or under any other patent rights owned or licensable by Applied Biosystems. Further information relating to the purchase of licenses for DNA sequence and fragment analysis and other applications may be obtained by contacting
the Director of Licensing at Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404, U.S.A.
Notice to Purchaser: Limited License
The purchase of the BigDye® Terminator v1.1 Cycle Sequencing Kit includes a limited, nontransferable, non-exclusive license
(without the right to resell, repackage, or sublicense) under the process claims of U.S. patents, and corresponding claims in foreign
counterpart patents and patent applications, to use this product solely with an Applied Biosystems commercial automated DNA
sequencing machine or other authorized automated DNA sequencing machines that have been authorized under these patents by
Applied Biosystems. No license is hereby granted for the use of this kit or the reagents therein in any other automated sequencing
machine. Such license is granted solely for research and other uses that are not unlawful. No other license is granted expressly,
impliedly, or by estoppel. For information concerning the availability of additional licenses to practice the patented
methodologies, contact: Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404,
USA. Patents are pending in countries outside the United States.
Notice to Purchaser About Limited License
This kit (reagent) is sold pursuant to a limited sublicense from Amersham International plc under one or more U.S. Patent Nos.
5,498,523 and 5,614,365 and corresponding foreign patents and patent applications. The purchase of this kit (reagent) includes a
limited non-exclusive sublicense (without the right to resell, repackage or further sublicense) under such patent rights to use this
reagent for DNA sequencing or fragment length analysis solely with an Applied Biosystems commercial automated sequencing
machine or other authorized DNA sequencing machines that have been authorized for such use by Applied Biosystems, or for
manual DNA sequencing. No license is hereby granted for use of this kit, or the reagents therein, in any other automated
sequencing machine. Such sublicense is granted solely for research or other uses that are not unlawful. No other license is
granted expressly, impliedly, or by estoppel. For information concerning the availability of additional license to practice the
patented methodologies, contact: Amersham Life Science, Inc., Vice President, Regulatory Affairs, P.O. Box 22400, Cleveland,
Ohio 44122. Patents are pending in countries outside the United States.
ABI PRISM, Applied Biosystems, BigDye, GeneScan, MicroAmp, and Primer Island are registered trademarks of Applied
Biosystems or its subsidiaries in the U.S. and certain other countries., and ABI, Avant, CATALYST, Hi-Di, POP, POP-4, POP-5,
and POP-6 are trademarks of Applied Biosystems or its subsidiaries in the U.S. and certain other countries..
Centricon is a trademark of W. R. Grace and Co.
Centri-Sep is a trademark of Princeton Separations, Inc.
GeneAmp are registered trademarks of Roche Molecular Systems, Inc.
pGEM is a registered trademark of Promega Corporation.
All other trademarks are the sole property of their respective owners.
Part Number 4337036 Rev. B
08/2010
Contents
Chapter 1
Introduction
Chapter Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1
In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1
About the Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
New Formulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Features and Compatibilities . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
BigDye Terminator v1.1 Cycle Sequencing Kit . . . . . . . . . . . . . . .
1-1
1-1
1-1
1-2
Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3
Instrument Platforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3
Thermal Cyclers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3
Required Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Dye/Filter Sets and Matrix Standards for the 310
and 377 Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Dye Sets and Spectral Standards for the 3700, 3100,
and 3100-Avant Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Instructions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Dye Set/Primer (Mobility) Files . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1-4
Reagents and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Available Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Kit Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Storage and Use of the Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1-6
1-6
1-6
1-7
1-4
1-4
1-5
1-6
Materials Supplied by the User . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-7
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-7
Materials for Cycle Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-8
Materials for Purifying Extension Products . . . . . . . . . . . . . . . . . . 1-9
Other Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-9
Materials for Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-10
General Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-11
BigDye® Terminator v1.1 Cycle Sequencing Kit
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August 21, 2002 8:36 am, 4337036A_v1.1TOC.fm
iii
Chapter 2
Preparing the Templates
Chapter Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1
In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1
Control DNA Templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Using Control DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Control DNA Sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
An Additional Control Sold Separately . . . . . . . . . . . . . . . . . . . . .
2-1
2-1
2-1
2-1
Template Preparation Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Single- and Double-Stranded Templates . . . . . . . . . . . . . . . . . . .
BAC DNA Templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
PCR Templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Purifying PCR Fragments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2-3
2-3
2-3
2-3
2-4
DNA Quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Poor Template Quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Determining DNA Quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2-4
2-4
2-5
2-5
DNA Quantity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-6
Quantitating DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-6
Template Quantity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-6
Chapter 3
Performing Cycle Sequencing
Chapter Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-1
In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-1
iv
Sequencing Single- and Double-Stranded DNA . . . . . . . . . . . . . . . . .
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Preparing the Reactions for 96-Well Reaction Plates
or Microcentrifuge Tubes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Using BigDye Terminator Sequencing Buffer . . . . . . . . . . . . . . . .
Preparing the Reactions for 384-Well Plates . . . . . . . . . . . . . . . . .
Cycle Sequencing on the System 9700, 9600, 2700, or 2400 . . .
3-2
3-3
3-4
3-5
Sequencing Large DNA Templates . . . . . . . . . . . . . . . . . . . . . . . . . . .
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Thermal Cyclers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Preparing Sequencing Reactions . . . . . . . . . . . . . . . . . . . . . . . . .
Performing Cycle Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3-5
3-5
3-5
3-6
3-7
DRAFT
3-2
3-2
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August 21, 2002 8:36 am, 4337036A_v1.1TOC.fm
Chapter 4
Purifying Extension Products
Chapter Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-1
In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-1
Choosing a Method of Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-1
Purpose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-1
Purification Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-1
Ethanol/EDTA Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Recommended Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Precipitating in 96-Well Reaction Plates . . . . . . . . . . . . . . . . . . . .
Precipitating in 384-Well Reaction Plates . . . . . . . . . . . . . . . . . . .
4-2
4-2
4-2
4-4
Ethanol/EDTA/Sodium Acetate Precipitation . . . . . . . . . . . . . . . . . . . . 4-6
Precipitating in 96-Well Reaction Plates . . . . . . . . . . . . . . . . . . . . 4-6
Precipitating in 384-Well Reaction Plates . . . . . . . . . . . . . . . . . . . 4-8
Plate and Spin Column Purification . . . . . . . . . . . . . . . . . . . . . . . . . .
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Recommended Spin Columns . . . . . . . . . . . . . . . . . . . . . . . . . . .
Optimizing Spin Column Purification . . . . . . . . . . . . . . . . . . . . . .
Performing Spin Column Purification . . . . . . . . . . . . . . . . . . . . . .
4-10
4-10
4-10
4-10
4-11
Plate Purification Using SDS/Heat Treatment . . . . . . . . . . . . . . . . . . . 4-13
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-13
Preparing Extension Products . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-13
Performing 96-Well Spin Plate Purification . . . . . . . . . . . . . . . . . . . . . 4-14
Recommended 96-Well Spin Plates . . . . . . . . . . . . . . . . . . . . . . . 4-14
Purifying with the 96-Well Spin Plate . . . . . . . . . . . . . . . . . . . . . . 4-14
Appendix A Selecting Sequencing Primers
Selecting Sequencing Primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-1
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-1
Optimizing Primer Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-1
Appendix B Control DNA Sequence
Control Sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-1
Partial Sequence of pGEM-3Zf(+) . . . . . . . . . . . . . . . . . . . . . . . . . B-1
BigDye® Terminator v1.1 Cycle Sequencing Kit
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v
Appendix C Sample Electrophoresis
Some Important Reminders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-1
Electrophoresis on the ABI PRISM 3700 DNA Analyzer . . . . . . . . . . . . C-2
Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-2
Performing Sample Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . C-3
Electrophoresis on the ABI PRISM 3100 and 3100-Avant
Genetic Analyzers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-4
Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-4
Performing Sample Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . C-5
Electrophoresis on the ABI PRISM 310 Genetic Analyzer . . . . . . . . . . . C-6
Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-6
Performing Sample Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . C-7
Electrophoresis on the ABI PRISM 377 DNA Sequencers . . . . . . . . . . .
Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Using the Lane Guide Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Using Long-Read Gel and Buffer Formulations . . . . . . . . . . . . . . .
Performing Sample Electrophoresis . . . . . . . . . . . . . . . . . . . . . . .
C-8
C-8
C-9
C-9
C-9
Appendix D Troubleshooting
Appendix E Obtaining Technical Support
Services and Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-1
Applied Biosystems Web Site . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-1
Index
vi
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Introduction
1
1
Chapter Summary
In This Chapter
The following topics are covered in this chapter:
About the Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-1
Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-3
Required Software. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-4
Reagents and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-6
Materials Supplied by the User . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-7
General Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-11
About the Kit
New Formulation
The BigDye® Terminator v1.1 Cycle Sequencing Kit has a new
formulation that delivers:
• Increased robustness
• High quality data
• Improved success with difficult templates
The protocols for cycle sequencing have been modified to optimize
results using the new formulation.
Features and
Compatibilities
• The BigDye Terminator v1.1 Cycle Sequencing Kit uses v1.0
matrix and spectral files and does not require new instrument
(matrix) files for the ABI PRISM® 310 Genetic Analyzer, and ABI
PRISM® 377 DNA Sequencers or new spectral calibrations for the
ABI PRISM ® 3700 DNA Analyzer, ABI PRISM ® 3100 Genetic
Analyzer, and ABI PRISM ® 3100-Avant Genetic Analyzer.
BigDye® Terminator v1.1 Cycle Sequencing Kit
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August 27, 2002 12:26 pm, 01_Introduction.fm
1-1
Chapter 1
Introduction
Applied Biosystems recommends that you verify the quality of
your current matrix before proceeding. If it is necessary to
generate a new matrix, use the appropriate matrix and/or
sequencing standard for your instrument.
– The 310 and 377 instruments use the 310/377 BigDye®
Terminator v1.1 Matrix Standards (PN 4336805) for instrument
(matrix) file generation.
– The 3700 instrument requires the 3700 BigDye® Terminator
v1.1 Matrix Standard (PN 4336825) for spectral calibration.
– The 3100 and 3100-Avant instruments require the 3100
BigDye® Terminator v1.1 Matrix Standard (PN 4336824) for
spectral calibration.
• The alcohol precipitation methods are different from those
recommended for the original and other versions of BigDye®
terminators.
• The existing mobility files can be used with their respective
platforms. New mobility files are not necessary.
BigDye
Terminator v1.1
Cycle
Sequencing Kit
The BigDye Terminator v1.1 Cycle Sequencing Kit provides the
required reagent components for the sequencing reaction in a ready
reaction, pre-mixed format. You need only provide your template and
template-specific primer.
These reagents are suitable for performing fluorescence-based cycle
sequencing reactions on single-stranded or double-stranded DNA
templates, on polymerase chain reaction (PCR) fragments, and on
large templates (for example, BAC clones).
Note: This kit includes BigDye® Terminator v1.1/3.1 Sequencing
Buffer (5X), which has been specifically optimized for use with the
new BigDye® ready reaction mixes. This buffer should be used for
any reaction optimization you undertake.
1-2
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August 27, 2002 12:26 pm, 01_Introduction.fm
Instruments
Instruments
Instrument
Platforms
The BigDye Terminator v1.1 Cycle Sequencing Kit is for use with
the following instruments:
•
•
•
•
•
ABI PRISM ® 3700 DNA Analyzer
ABI PRISM ® 3100 Genetic Analyzer
ABI PRISM ® 3100-Avant Genetic Analyzer
ABI PRISM ® 310 Genetic Analyzer
ABI PRISM® 377 DNA Sequencer (all models*)
General instructions are given for using the kit reagents to generate
samples for these instruments. For more detailed instructions, refer to
the appropriate instrument user’s manual or chemistry guide.
Thermal Cyclers
The protocols provided in this document were optimized using
Applied Biosystems thermal cyclers, including the GeneAmp® PCR
Systems 9700, 9600, 2700, and 2400.
Note: If you use a thermal cycler not manufactured by Applied
Biosystems, you may need to optimize thermal cycling conditions.
Ramping time is very important. If the thermal ramping time is too
fast (>1°/sec), poor (noisy) data may result.
*Includes the ABI PRISM 377, ABI PRISM 377-18, ABI PRISM 377 with XL
Upgrade, and the ABI PRISM 377 with 96-Lane Upgrade instruments.
BigDye® Terminator v1.1 Cycle Sequencing Kit
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1-3
Chapter 1
Introduction
Required Software
Dye/Filter Sets
and Matrix
Standards for the
310 and 377
Instruments
The dye/filter sets and matrix standards required for the 310 and 377
instruments are listed in the table below.
Instrument
Dye/Filter Set
310 Genetic Analyzer
Filter Set E
377 DNA Sequencer†
Filter Set E
Standards for
Instrument (Matrix)
File Generation
310/377 BigDye
Terminator v1.1 Matrix
Standards
(PN 4336805)*
*The BigDye Terminator v1.1 Cycle Sequencing Kit does not require a new
spectral calibration file if you currently have a BigDye® Terminator v1.0
spectral file on your instrument.
†Includes the ABI PRISM 377, ABI PRISM 377-18, ABI PRISM 377 with XL
Upgrade, and the ABI PRISM 377 with 96-Lane Upgrade instruments.
Dye Sets and
Spectral
Standards for the
3700, 3100, and
3100-Avant
Instruments
IMPORTANT! Spectral calibrations for the BigDye terminators v1.1
are not compatible with the v3.0 or v3.1 terminators.
Instrument
Dye/Filter Set
3700 DNA Analyzer
Filter Set E
3100 Genetic Analyzer
3100-Avant Genetic
Analyzer
Filter Set E
Standards for
Instrument (Matrix)
File Generation
3700 BigDye
Terminator v1.1 Matrix
Standard
(PN 4336825)*
3100 BigDye®
Terminator v1.1 Matrix
Standard
(PN 4336824)*
*The BigDye Terminator v1.1 Cycle Sequencing Kit does not require a new
spectral calibration file if you currently have a BigDye Terminator v1.0
spectral file on your instrument.
IMPORTANT! Users of the ABI Prism 3700 DNA Analyzer refer to
the ABI Prism 3700 DNA Analyzer User’s Manual (PN 4306152) for
information on instrument (matrix) files.
1-4
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Required Software
Instructions
For Generating Matrices
For the 377 and 310 instruments, refer to the product insert (included
with matrix or sequence standards) for instructions on using the
BigDye Matrix Standards v1.1 to generate matrices.
For Performing Spectral Calibrations
• For the 3700 instrument, refer to the product insert for instructions
on using the 3700/3730 BigDye Terminator v1.1 Matrix Standard
to perform spectral calibration.
• For the 3100 and 3100-Avant instruments, refer to the product
insert for instructions on using the 3100 BigDye Terminator v1.1
Matrix Standard to perform spectral calibration.
BigDye® Terminator v1.1 Cycle Sequencing Kit
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August 27, 2002 12:26 pm, 01_Introduction.fm
1-5
Chapter 1
Introduction
Dye Set/Primer
(Mobility) Files
To analyze sequencing data generated with BigDye® chemistries
v1.1, you need dye set/primer (mobility) files that were created for
v1.0 chemistries. The dye set/primer (mobility) files can be
downloaded from the Internet.
Dye set/primer (mobility) files can be downloaded from the
Applied Biosystems website:
http://www.appliedbiosystems.com/support/software
If you do not have access to the Internet, contact Applied Biosystems
Technical Support, or your local field applications specialist (call
your local sales office for more information).
Reagents and Storage
Available Kits
The following kits are available:
Number of
Reactions
Kit
BigDye Terminator v1.1 Cycle
Sequencing Kit
Part Number
100
4337450
1000
4337451
5000
4337452
25,000
4337453
The BigDye® Terminator v1.1 Cycle Sequencing Kit Protocol
(PN 4337036) is available separately and can be ordered at no charge.
Kit Reagents
A listing of the kit components is given below.
•
•
•
•
1-6
Ready Reaction Mix
pGEM®-3Zf(+) double-stranded DNA Control Template
–21 M13 Control Primer (forward)
BigDye Terminator v1.1/3.1 Sequencing Buffer (5X)
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Materials Supplied by the User
Storage and Use
of the Kits
• Store the kit at –15 to –25 °C.
Note: The BigDye sequencing buffer can be stored at 4 °C.
• Avoid excess (that is, no more than 5 to 10) freeze-thaw cycles.
Aliquot reagents in smaller amounts if necessary.
• Before each use of the kit, allow the frozen stocks to thaw at room
temperature (do not heat).
IMPORTANT! Mix each stock thoroughly and then centrifuge
briefly to collect all the liquid at the bottom of each tube.
• Whenever possible, keep thawed materials on ice during use. Do
not leave reagents at room temperature for extended periods.
Materials Supplied by the User
Overview
In addition to the reagents supplied in this kit, other items are
required.
This section lists general materials needed for:
• Cycle sequencing
• Purifying extension products
Note: Many of the items listed in this section are available from
major laboratory suppliers (MLS) unless otherwise noted. Equivalent
sources may be acceptable where noted.
Refer to the individual instrument protocols for the specific items
needed for each instrument.
CHEMICAL HAZARD. Before handling the
chemical reagents needed for cycle sequencing, read the safety
warnings on the reagent bottles and in the manufacturers’ Material
Safety Data Sheets (MSDSs), and follow the handling instructions.
Wear appropriate protective eyewear, clothing, and gloves. Dispose of
waste in accordance with all local, state/provincial, and national
environmental and health regulations.
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1-7
Chapter 1
Introduction
Materials for
Cycle
Sequencing
The table below lists the plates or tubes required for the
recommended Applied Biosystems thermal cyclers.
Thermal Cycler
Plate or Tube
Applied Biosystems
Part Number
GeneAmp® PCR System
9700
MicroAmp® 384-Well Reaction Plate
4305505
MicroAmp® 96-Well Reaction Plate
N801-0560
MicroAmp® Reaction Tubes, 0.2-µL
N801-0533
MicroAmp® Caps, 12 or 8/strip
N801-0534 or N8010535
ABI PRISM™ Optical Adhesive Cover
Starter Pack or ABI PRISM ® Optical
Adhesive Covers
4313663 or 4311971
MicroAmp® 96-Well Reaction Plate
N801-0560
MicroAmp® Reaction Tubes, 0.2-µL
N801-0533
MicroAmp® Caps, 12 or 8/strip
N801-0534 N801-0535
ABI PRISM™ Optical Adhesive Cover
Starter Pack or ABI PRISM ® Optical
Adhesive Covers
4313663 or 4311971
MicroAmp® Reaction Tubes, 0.2-mL
N801-0533
MicroAmp® Caps, 12 or 8/strip
N801-0534 N801-0535
GeneAmp® PCR System
9600
GeneAmp® PCR System
2400 and 2700
1-8
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Materials Supplied by the User
Materials for
Purifying
Extension
Products
Method
Material
Supplier
Ethanol/EDTA
Precipitation
Ethanol (EtOH), 200
proof, Molecular Biology
grade
MLS
EDTA, 125 mM
Sealing taped
Ethanol/Sodium
Acetate
Precipitation
Ethanol (EtOH), 200
proof, Molecular Biology
grade
Sodium acetate
(NaOAc), 3 M, pH 5.2
Sealing tape
Plate Column
Purification
Note: For 96-well
reaction plates
Spin Column
Purification
Other Equipment
96-Well columns for
purification
Sealing tape
MLS
Costar 6570
Thermowell Sealing
Tape
MLS
Applied Biosystems
(PN 400320)
Costar 6570
Thermowell Sealing
Tape
See “Recommended
96-Well Spin Plates”
on page 4-14
Costar 6570
Thermowell Sealing
Tape
Centri-Sep™ spin
column, 1-mL, 32
columns, 100 columns
Applied Biosystems
PN 401763,
PN 401762
Sealing tape
Costar 6570
Thermowell Sealing
Tape
You will also need a variable speed centrifuge with microtiter plate
holders capable of reach a spin speed of at least 1400 × g.
Applied Biosystems recommends a Beckman Allegra 6A centrifuge
with a GH-3.8A rotor.
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1-9
Chapter 1
Introduction
Materials for
Electrophoresis
Instrument
Material
Supplier
ABI PRISM 3700
DNA Analyzer
Hi-Di™ Formamide, 25-mL
bottle
Applied Biosystems
(PN 4311320)
3700 BigDye Terminator
v1.1 Matrix Standard
Applied Biosystems
(PN 4336825)
3700/3730 BigDye®
Terminator v1.1
Sequencing Standard
Applied Biosystems
(PN 4336799)
Hi-Di™ Formamide, 25-mL
bottle
Applied Biosystems
(PN 4311320)
3100 BigDye Terminator
v1.1 Matrix Standard
Applied Biosystems
(PN 4336824)
BigDye® Terminator v1.1
Sequencing Standard
Applied Biosystems
(PN 4336791)
Template Suppression
Reagent (TSR)
Applied Biosystems
(PN 401674)
EDTA
MLS
310/377 BigDye
Terminator v1.1 Matrix
Standards
Applied Biosystems
(PN 4336805)
Formamide
MLS
EDTA
MLS
25 mM EDTA with
50 mg/mL blue dextran
Applied Biosystems
(PN 402055)
310/377 BigDye
Terminator v1.1 Matrix
Standards
Applied Biosystems
(PN 4336805)
ABI PRISM 3100
and 3100-Avant
Genetic Analyzers
ABI PRISM 310
Genetic Analyzer
ABI PRISM 377 DNA
Sequencer
1-10
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General Safety
General Safety
Documentation
User Attention
Words
Five user attention words appear in the text of all Applied Biosystems
user documentation. Each word implies a particular level of
observation or action as described below.
Note: Calls attention to useful information.
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 which,
if not avoided, may result in minor or moderate injury. It may also be
used to alert against unsafe practices.
Indicates a potentially hazardous situation which,
if not avoided, could result in death or serious injury.
Indicates an imminently hazardous situation
which, if not avoided, will result in death or serious injury. This
signal word is to be limited to the most extreme situations.
Site Preparation
and Safety Guide
A site preparation and safety guide is a separate document sent to all
customers who have purchased an Applied Biosystems instrument.
Refer to the guide written for your instrument for information on site
preparation, instrument safety, chemical safety, and waste profiles.
Chemical Safety
Chemical Hazard
Warning
CHEMICAL HAZARD. Some of the chemicals
used with Applied Biosystems instruments and protocols are
potentially hazardous and can cause injury, illness, or death.
• Read and understand the material safety data sheets (MSDSs)
provided by the chemical manufacturer before you store, handle,
or work with any chemicals or hazardous materials.
• Minimize contact with chemicals. Wear appropriate personal
protective equipment when handling chemicals (e.g., safety
glasses, gloves, or protective clothing). For additional safety
guidelines, consult the MSDS.
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1-11
Chapter 1
Introduction
• Minimize the inhalation of chemicals. Do not leave chemical
containers open. Use only with adequate ventilation (e.g., fume
hood). For additional safety guidelines, consult the MSDS.
• Check regularly for chemical leaks or spills. If a leak or spill
occurs, follow the manufacturer’s cleanup procedures as
recommended on the MSDS.
• Comply with all local, state/provincial, or national laws and
regulations related to chemical storage, handling, and disposal.
Chemical Waste
Hazard Warning
CHEMICAL WASTE HAZARD. Wastes
produced by Applied Biosystems instruments are potentially
hazardous and can cause injury, illness, or death.
• Read and understand the material safety data sheets (MSDSs)
provided by the manufacturers of the chemicals in the waste
container before you store, handle, or dispose of chemical waste.
• Handle chemical wastes in a fume hood.
• Minimize contact with chemicals. Wear appropriate personal
protective equipment when handling chemicals (e.g., safety
glasses, gloves, or 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 (e.g., fume
hood). For additional safety guidelines, consult the MSDS.
• After emptying the waste container, seal it with the cap provided.
• Dispose of the contents of the waste tray and waste bottle in
accordance with good laboratory practices and local,
state/provincial, or national environmental and health regulations.
About MSDSs
Some of the chemicals used with this instrument may be listed as
hazardous by their manufacturer. When hazards exist, warnings are
prominently displayed on the labels of all chemicals.
Chemical manufacturers supply a current material safety data sheet
(MSDS) before or with shipments of hazardous chemicals to new
customers and with the first shipment of a hazardous chemical after
an MSDS update. MSDSs provide you with the safety information
you need to store, handle, transport and dispose of the chemicals
safely.
1-12
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Chemical Safety
We strongly recommend that you replace the appropriate MSDS in
your files each time you receive a new MSDS packaged with a
hazardous chemical.
CHEMICAL HAZARD. Be sure to familiarize
yourself with the MSDSs before using reagents or solvents.
Ordering MSDSs
You can order free additional copies of MSDSs for chemicals
manufactured or distributed by Applied Biosystems using the contact
information below.
To order documents by automated telephone service:
1.
From the U.S. or Canada, dial 1.800.487.6809.
2.
Follow the voice instructions to order documents (for
delivery by fax).
Note: There is a limit of five documents per fax request.
To obtain documents through the Applied Biosystems Web site:
1. Go to
http://docs.appliedbiosystems.com/msdssearch.html
2. In the SEARCH field, type in the chemical name, part
number, or other information that will appear in the MSDS
and click SEARCH.
Note: You may also select the language of your choice from
the drop-down list.
3. When the Search Results page opens, find the document
you want and click on it to open a PDF of the document.
For chemicals not manufactured or distributed by
Applied Biosystems, call the chemical manufacturer.
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1-13
Chapter 1
1-14
Introduction
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Preparing the Templates
2
2
Chapter Summary
In This Chapter
The following topics are covered in this chapter:
Control DNA Templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-1
Template Preparation Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-3
DNA Quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-4
DNA Quantity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-6
Control DNA Templates
Using Control
DNA
Include a control DNA template as one of the templates in a set of
sequencing reactions. The results from the control can help determine
whether failed reactions are the result of poor template quality or
sequencing reaction failure.
Control DNA
Sequence
Applied Biosystems recommends M13mp18 as a single-stranded
control and pGEM®-3Zf(+) as a double-stranded control. All Applied
Biosystems DNA sequencing kits provide pGEM® control DNA. All
dye terminator cycle sequencing kits include a –21 M13 forward
primer for use in performing control reactions.
The partial sequence of pGEM-3Zf(+) from the –21 M13 forward
primer, followed by the ensuing 1000 bases is shown in Appendix A,
“Control DNA Sequence.”
An Additional
Control Sold
Separately
The BigDye® Terminator v1.1 Sequencing Standard provides an
additional control to help in troubleshooting electrophoresis runs.
This standard contains lyophilized sequencing reactions that require
only resuspension and denaturation before use.
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2-1
Chapter 2
Preparing the Templates
There are two forms of the v1.0 and v1.1 sequencing standard, as
shown in the table below. Please use the correct sequencing standard
for your instrument. Refer to the product inserts for instructions on
using each sequencing standard.
Instrument
Kit
PN
ABI PRISM ® 3700 DNA Analyzer
3700/3730 BigDye®
Terminator v1.1
Sequencing Standard
4336799
BigDye® Terminator
v1.1 Sequencing
Standard
4336791
ABI PRISM ® 3100 Genetic Analyzer
ABI PRISM ® 3100-Avant Genetic
Analyzer
ABI PRISM 310 Genetic Analyzer
®
ABI PRISM ® 377 DNA Sequencers*
*Includes the ABI PRISM 377, ABI PRISM 377-18, ABI PRISM 377 with XL
Upgrade, and the ABI PRISM 377 with 96-Lane Upgrade instruments.
2-2
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Template Preparation Methods
Template Preparation Methods
Single- and
Double-Stranded
Templates
BAC DNA
Templates
Refer to the Automated DNA Sequencing Chemistry Guide
(PN 4305080) for information on preparing single- and doublestranded templates.
With larger DNA targets such as bacterial artificial chromosomes
(BACs), the quality of DNA template is important to the success of
the sequencing reaction. Two methods have given good sequencing
results:
• Alkaline lysis,* with extra phenol extraction and isopropanol
precipitation if very clean DNA is desired
• Cesium chloride (CsCl) banding
Commercial Kits
Commercial kits are also available for BAC DNA preparation:
• QIAGEN-tip 100 (QIAGEN: PN 10043, 25 reactions; 10045,
100 reactions)
• QIAGEN-tip 500 (QIAGEN: PN 10063, 25 reactions; 10065,
100 reactions)
PCR Templates
Cycle sequencing provides the most reproducible results for
sequencing PCR templates. Although PCR fragments can be difficult
to denature with traditional sequencing methods, cycle sequencing
provides several chances to denature and extend the template,
ensuring adequate signal in the sequencing reaction.
Importance of Purifying Product
For optimum results, purify the PCR product before sequencing. In
general, any method that removes dNTPs and primers should work.
We recommend Centricon®-100 columns (PN N930-2119). The
protocol for using these columns is provided in “Purifying PCR
Fragments” on page 2-4
Refer to the Automated DNA Sequencing Chemistry Guide for
information on sequencing PCR templates.
*Marra, M., Weinstock, L.A., and Mardis, E.R. 1966. End sequence
determination from large insert cloning using energy transfer fluorescent
primers. Genomic Methods 6: 1118-1122.
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2-3
Chapter 2
Preparing the Templates
Purifying PCR
Fragments
To purify PCR fragments by ultrafiltration:
1. Assemble the Centricon-100 column according to the
manufacturer’s recommendations.
2. Load 2 mL deionized water onto the column.
3. Add the entire sample to the column.
4. Spin the column at 3000 × g in a fixed-angle centrifuge for
10 minutes.
Note: The manufacturer recommends a maximum speed of
1000 × g, but 3000 × g has worked well in Applied
Biosystems laboratories. If you are following the
manufacturer’s guidelines, increase the time to compensate.
5. Remove the waste receptacle and attach the collection vial.
6. Invert the column and spin it at 270 × g for 2 minutes to
collect approximately 40–60 µL of sample.
7. Add deionized water to bring the purified PCR fragments to
the original volume.
DNA Quality
Poor Template
Quality
Poor template quality is the most common cause of sequencing
problems. The following are characteristics of poor quality templates:
• Noisy data or peaks under peaks
• No usable sequence data
• Weak signal
Always follow recommended procedures to prepare templates.
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DNA Quality
Contamination
Potential contaminants include:
•
•
•
•
Proteins
RNA
Chromosomal DNA
Excess PCR primers, dNTPs, enzyme, and buffer components
(from a PCR amplification used to generate the sequencing
template)
• Residual salts
• Residual organic chemicals such as phenol, chloroform, and
ethanol
• Residual detergents
Determining DNA
Quality
The following methods can be used to examine DNA quality:
• Agarose gel electrophoresis
Purified DNA should run as a single band on an agarose gel.
Note: Uncut plasmid DNA can run as three bands: supercoiled,
nicked, and linear.
• Spectrophotometry
The A260/A280 ratio should be 1.7 to 1.9. Smaller ratios usually
indicate contamination by protein or organic chemicals.
Agarose gels reveal the presence of contaminating DNAs and RNAs,
but not proteins. Spectrophotometry can indicate the presence of
protein contamination, but not DNA and RNA contamination. Use
these methods together to get the most information about your DNA
before sequencing.
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2-5
Chapter 2
Preparing the Templates
DNA Quantity
Quantitating DNA
Template
Quantity
If possible, quantitate the amount of purified DNA by measuring the
absorbance at 260 nm or by some other method.
The table below shows the amount of template to use in a cycle
sequencing reaction.
Template
Quantity
PCR product:
100–200 bp
200–500 bp
500–1000 bp
1000–2000 bp
>2000 bp
1–3 ng
3–10 ng
5–20 ng
10–40 ng
20–50 ng
Single-stranded
25–50 ng
Double-stranded
150–300 ng
Cosmid, BAC
0.5–1.0 µg
Bacterial genomic DNA
2–3 µg
Note: In general, higher DNA quantities give higher signal
intensities. Higher DNA quantities may also give shorter read lengths
and top-heavy data.
The template quantities given above should work with all primers.
You may be able to use even less DNA when using capillary
instruments for detection. The amount of PCR product to use in
sequencing also depends on the length and purity of the PCR product.
2-6
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Performing Cycle Sequencing 3
3
Chapter Summary
In This Chapter
The following topics are covered in this chapter:
Sequencing Single- and Double-Stranded DNA . . . . . . . . . . . . . . .3-2
Sequencing Large DNA Templates . . . . . . . . . . . . . . . . . . . . . . . . .3-5
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3-1
Chapter 3
Performing Cycle Sequencing
Sequencing Single- and Double-Stranded DNA
Overview
Preparing the
Reactions for 96Well Reaction
Plates or
Microcentrifuge
Tubes
This section describes how to prepare reactions and perform cycle
sequencing on a variety of templates, including M13, plasmids, and
PCR products.
The type of tube required depends on the thermal cycler that you are
using. Refer to “Materials for Cycle Sequencing” on page 1-8.
To prepare the reaction mixtures:
1. For each reaction add the following reagents to a separate
tube:
Reagent
Quantity
Terminator Ready Reaction
Mix*
8.0 µL
Template
See the table in “Template
Quantity” on page 2-6.
Primer
3.2 pmol
Deionized water
q.s.
Total Volume
20 µL
2. Mix well and spin briefly.
*See “Using BigDye Terminator Sequencing Buffer” below.
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Sequencing Single- and Double-Stranded DNA
Using BigDye
Terminator
Sequencing
Buffer
The BigDye® Terminator v1.1/3.1 Sequencing Buffer (5X)* is
supplied at a 5X concentration. If you use it for sequencing reactions,
be sure the final reaction volume is at a concentration of 1X. For
example, for a half reaction in 20 µL final volume, you would use
4 µL of ready reaction premix and 2 µL of BigDye sequencing buffer
as shown below.
Reagent
Concentration
Volume
2.5X
4 µL
BigDye Sequencing
Buffer
5X
2 µL
Primer
—
3.2 pmol
Template
—
See
“Template
Quantity” on
page 2-6.
Water
—
to 20 µL
Final Volume
1X
20 µL
Ready Reaction Premix
Note: The use of this buffer without optimization may result in
deterioration of sequence quality. Applied Biosystems does not
support diluted reactions or guarantee the performance of BigDye®
chemistry when it is diluted.
*The BigDye Terminator v1.1/3.1 Sequencing Buffer is intended for use only
with BigDye Terminator v1.1/3.l Cycle Sequencing Kits.
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3-3
Chapter 3
Performing Cycle Sequencing
Preparing the
Reactions for
384-Well Plates
The type of tube required depends on the thermal cycler that you are
using. Refer to “Materials for Cycle Sequencing” on page 1-8.
Note: The wells in a 384-well reaction plate have a volume capacity
of 35 µL. Therefore, Applied Biosystems recommends doing a 10 µL
reaction. This allows the post-reaction cleanup step to be performed
in the same well.
To prepare the reaction mixtures:
1. For each reaction add the following reagents to a separate
tube:
Reagent
Quantity
Terminator Ready Reaction
Mix*
4.0 µL
Template
See the table in “Template
Quantity” on page 2-6.
Primer
3.2 pmol
Deionized water
q.s.
Total Volume
10 µL
2. Mix well and spin briefly.
3. Use on a GeneAmp® PCR System 9700 Dual 384-Well
Sample Block Module.
*Note: For instructions on using BigDye sequencing buffer, see “Using
BigDye Terminator Sequencing Buffer” on page 3-3.
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Sequencing Large DNA Templates
Cycle
Sequencing on
the System 9700,
9600, 2700, or
2400
To sequence single- and double-stranded DNA on the GeneAmp®
PCR System 9700 (in 9600 emulation mode), 9600, 2700, or 2400:
1. Place the tubes in a thermal cycler and set to the correct
volume.
2. Perform an initial denaturation.
a. Rapid thermal ramp to 96 °C
b. 96 °C for 1 min
3. Repeat the following for 25 cycles:
• Rapid thermal ramp* to 96 °C
• 96 °C for 10 seconds.
• Rapid thermal ramp to 50 °C
• 50 °C for 5 seconds.
• Rapid thermal ramp to 60 °C
• 60 °C for 4 minutes
4. Rapid thermal ramp to 4 °C and hold until ready to purify.
5. Spin down the contents of the tubes in a microcentrifuge.
6. Proceed to Chapter 4, “Purifying Extension Products.”
*Rapid thermal ramp is 1 °C/second.
Sequencing Large DNA Templates
Overview
This section describes how to prepare reactions and perform cycle
sequencing on large DNA templates such as:
• BAC DNA
• Cosmid DNA
• Genomic DNA
Thermal Cyclers
Only the following thermal cyclers can be used with this protocol:
• GeneAmp PCR System 9600
• GeneAmp PCR System 9700 (in 9600 emulation mode)
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3-5
Chapter 3
Performing Cycle Sequencing
Reoptimize this protocol for use on other thermal cyclers.
Preparing
Sequencing
Reactions
The type of tube required depends on the thermal cycler that you are
using. Refer to “Materials Supplied by the User” on page 1-7.
To prepare the sequencing reaction:
1. For each reaction, add the following reagents to a separate
tube:
Reagent
Quantity
Terminator Ready Reaction Mix*
8.0 µL
DNA Template
See the table in
“DNA Quantity” on
page 2-6
Primer
3.2 pmol
Deionized water
q.s.
Total Volume
20 µL
2. Mix well and spin briefly.
*Note: For instructions on using BigDye sequencing buffer, see “Using
BigDye Terminator Sequencing Buffer” on page 3-3.
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Sequencing Large DNA Templates
Performing Cycle
Sequencing
To perform cycle sequencing on BAC DNA:
1. Place the tubes in a thermal cycler and set the volume to
20 µL.
2. Heat the tubes at 95 °C for 5 minutes.
3. Repeat the following for 30 cycles:*
• Rapid thermal ramp† to 95 °C
• 95 °C for 30 seconds.
• Rapid thermal ramp to 50–55 °C (depending on template)
• 50–55 °C for 10 seconds.
• Rapid thermal ramp to 60 °C
• 60 °C for 4 minutes.
4. Rapid thermal ramp to 4 °C and hold until ready to purify.
5. Spin down the contents of the tubes in a microcentrifuge.
6. Proceed to Chapter 4, “Purifying Extension Products.”
*Some laboratories have found that increasing the number of cycles gives
better results.
†Rapid thermal ramp is 1 °C/sec.
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3-7
Chapter 3
3-8
Performing Cycle Sequencing
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Purifying Extension Products 4
4
Chapter Summary
In This Chapter
The following topics are covered in this chapter:
Choosing a Method of Purification. . . . . . . . . . . . . . . . . . . . . . . . .4-1
Ethanol/EDTA Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-2
Ethanol/EDTA/Sodium Acetate Precipitation. . . . . . . . . . . . . . . . .4-6
Plate and Spin Column Purification . . . . . . . . . . . . . . . . . . . . . . .4-10
Plate Purification Using SDS/Heat Treatment . . . . . . . . . . . . . . .4-13
Performing 96-Well Spin Plate Purification . . . . . . . . . . . . . . . . .4-14
Choosing a Method of Purification
Purpose
The best results are obtained when unincorporated dye terminators
are completely removed prior to electrophoresis. Excess dye
terminators in sequencing reactions obscure data in the early part of
the sequence and can interfere with basecalling.
Purification
Methods
The methods recommended below have produced clean sequencing
data for BigDye® Terminator v1.1 Cycle Sequencing Kit products:
• Ethanol/EDTA precipitation
• Ethanol/EDTA/sodium acetate precipitation
• Plate and Spin column purification
Earlier methods developed for other sequencing kits may not remove
unincorporated dyes efficiently.
Use the method that works best for your particular application.
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4-1
Chapter 4
Purifying Extension Products
Note: These precipitation protocols have been optimized for use with
the v1.1 formulation at the specified sequencing reaction volumes
(20 µL in 96-well format and 10 µL in 384-well format) and are not
recommended for other versions.
IMPORTANT! To clean up sequencing reactions at volumes less than
those specified, reduce each component of the precipitation protocol
proportionately.
Ethanol/EDTA Precipitation
Recommended
Protocol
With the BigDye® terminators v1.1, the ethanol/EDTA precipitation
method produces consistent signal, while minimizing unincorporated
dyes. It is particularly good at getting rid of unincorporated dyelabelled terminators that obscure data in the early part of the
sequence.
Note: While this method produces the cleanest signal, it may cause
loss of small molecular weight fragments.
IMPORTANT! Absolute ethanol absorbs water from the atmosphere,
gradually decreasing its concentration and leading to inaccurate final
concentrations of ethanol, which can affect some sequencing results.
IMPORTANT! 95% ethanol is usable, but you must make sure the
final ethanol concentration for precipitation remains the same
(67–71%).
Precipitating in
96-Well Reaction
Plates
CHEMICAL HAZARD. EDTA. Exposure
causes eye irritation. Read the MSDS, and follow the handling
instructions. Wear appropriate protective eyewear, clothing, and
gloves.
CHEMICAL HAZARD. Ethanol is a
flammable liquid and vapor. Exposure causes eye, skin, and
respiratory tract irritation and may cause central nervous system
depression and liver damage. Read the MSDS, and follow the
handling instructions. Wear appropriate protective eyewear, clothing,
and gloves.
4-2
DRAFT
BigDye® Terminator v1.1 Cycle Sequencing Kit
August 27, 2002 9:57 am, 04_PurifyExtension.fm
Ethanol/EDTA Precipitation
To precipitate 20 µL sequencing reactions in 96-well reaction
plates:
1. Remove the 96-well reaction plate from the thermal cycler
and briefly spin.
2. Add 5 µL of 125 mM EDTA to each well.
Note: Make sure the EDTA reaches the bottom of the wells.
3. Add 60 µL of 100% ethanol to each well.
4. Seal the plate with aluminum tape and mix by inverting
4 times.
5. Incubate at room temperature for 15 min.
6.
If you are using ...
Then ...
a Beckman Allegra
6A centrifuge with a
GH-3.8A rotor
set it at 4 °C and spin the plate at
1650 × g for 45 min.
any other centrifuge
use a plate adapter and spin the plate
at the maximum speed as follows:
• 1400–2000 × g for 45 min
or
• 2000–3000 × g for 30 min
IMPORTANT! Proceed to the next step immediately. If this is
not possible, then spin the plate for an additional 2 min
before performing the next step.
7. Invert the plate and spin up to 185 × g, then remove from the
centrifuge.
8. Add 60 µL of 70% ethanol to each well.
9. With the centrifuge set to 4 °C, spin at 1650 × g for 15 min.
10. Invert the plate and spin up to 185 × g for 1 min, then remove
from the centrifuge.
Note: Start timing when the rotor starts moving.
BigDye® Terminator v1.1 Cycle Sequencing Kit
DRAFT
August 27, 2002 9:57 am, 04_PurifyExtension.fm
4-3
Chapter 4
Purifying Extension Products
To precipitate 20 µL sequencing reactions in 96-well reaction
plates: (continued)
11. To continue, resuspend the samples in injection buffer.
To store, cover with aluminum foil, and store at 4 °C.
Note: Make sure the wells are dry. You may use a Speed-Vac
for 15 min to dry the plate.
IMPORTANT! Make sure the samples are protected from
light while they are drying.
Precipitating in
384-Well
Reaction Plates
CHEMICAL HAZARD. EDTA. Exposure
causes eye irritation. Read the MSDS, and follow the handling
instructions. Wear appropriate protective eyewear, clothing, and
gloves.
CHEMICAL HAZARD. Ethanol is a
flammable liquid and vapor. Exposure causes eye, skin, and
respiratory tract irritation and may cause central nervous system
depression and liver damage. Read the MSDS, and follow the
handling instructions. Wear appropriate protective eyewear, clothing,
and gloves.
To precipitate in 384-well reaction plates:
1. Remove the 384-well reaction plates from the thermal cycler.
Remove the seal from each plate and briefly spin the plates.
2. Add 2.5 µL of 125 mM EDTA to each well.
Note: Make sure the EDTA reaches the bottom of the wells.
3. Add 25 µL of 100% ethanol to each well.
4. Seal the plates with aluminum tape and mix by inverting 4
times.
5. Incubate at room temperature for 15 min.
4-4
DRAFT
BigDye® Terminator v1.1 Cycle Sequencing Kit
August 27, 2002 9:57 am, 04_PurifyExtension.fm
Ethanol/EDTA Precipitation
To precipitate in 384-well reaction plates: (continued)
6.
If you are using ...
Then ...
a Beckman Allegra
6A centrifuge with a
GH-3.8A rotor
set it at 4 °C and spin the plate at
1650 × g for 45 min.
any other centrifuge
use a plate adapter and spin the plate
at the maximum speed as follows:
• 1400–2000 × g for 45 min
or
• 2000–3000 × g for 30 min
IMPORTANT! Proceed to the next step immediately. If this is
not possible, then spin the plate for an additional 2 min
before performing the next step.
7. Invert the plate and spin up to 185 × g, then remove from the
centrifuge.
8. Add 30 µL of 70% ethanol to each well.
9. With the centrifuge set to 4 °C, spin at 1650 × g for 15 min.
10. Invert the plate and spin up to 185 × g for 1 min, then remove
from the centrifuge.
Note: Start timing when the rotor starts moving.
11. To continue, resuspend the samples in injection buffer.
To store, cover with aluminum foil, and store at 4 °C.
Note: Make sure the wells are dry. You may use a Speed-Vac
for 15 min to dry the plate.
IMPORTANT! Make sure the samples are protected from
light while they are drying.
BigDye® Terminator v1.1 Cycle Sequencing Kit
DRAFT
August 27, 2002 9:57 am, 04_PurifyExtension.fm
4-5
Chapter 4
Purifying Extension Products
Ethanol/EDTA/Sodium Acetate Precipitation
Note: Ethano/EDTA/sodium acetate precipitation is recommended
when good signal from base 1 is required. However, for reactions
containing high concentrations of unincorporated terminators, some
residual terminators may be carried through the precipitation. To
completely remove excess terminators in these cases, ethanol/EDTA
precipitation is recommended (see page 4-2).
Precipitating in
96-Well Reaction
Plates
CHEMICAL HAZARD. EDTA. Exposure
causes eye irritation. Read the MSDS, and follow the handling
instructions. Wear appropriate protective eyewear, clothing, and
gloves.
CHEMICAL HAZARD. Ethanol is a
flammable liquid and vapor. Exposure causes eye, skin, and
respiratory tract irritation and may cause central nervous system
depression and liver damage. Read the MSDS, and follow the
handling instructions. Wear appropriate protective eyewear, clothing,
and gloves.
To precipitate 20 µL sequencing reactions in 96-well reaction
plates:
1. Remove the 96-well reaction plate from the thermal cycler
and briefly spin.
2. Add 2 µL of 125 mM EDTA to each well.
Note: Make sure the EDTA reaches the bottom of the wells.
3. Add 2 µL of 3 M sodium acetate to each well.
Note: Make sure the sodium acetate reaches the bottom of
the wells.
4. Add 50 µL of 100% ethanol to each well.
5. Seal the plate with aluminum tape and mix by inverting
4 times.
6. Incubate at room temperature for 15 min.
4-6
DRAFT
BigDye® Terminator v1.1 Cycle Sequencing Kit
August 27, 2002 9:57 am, 04_PurifyExtension.fm
Ethanol/EDTA/Sodium Acetate Precipitation
To precipitate 20 µL sequencing reactions in 96-well reaction
plates: (continued)
7.
If you are using ...
Then ...
a Beckman Allegra
6A centrifuge with a
GH-3.8A rotor
set it at 4 °C and spin the plate at
1650 × g for 45 min.
any other centrifuge
use a plate adapter and spin the plate
at the maximum speed as follows:
• 1400–2000 × g for 45 min
or
• 2000–3000 × g for 30 min
IMPORTANT! Proceed to the next step immediately. If this is
not possible, then spin the plate for an additional 2 min
before performing the next step.
8. Invert the plate and spin up to 185 × g, then remove from the
centrifuge.
9. Add 70 µL of 70% ethanol to each well.
10. With the centrifuge set to 4 °C, spin at 1650 × g for 15 min.
11. Invert the plate and spin up to 185 × g for 1 min, then remove
from the centrifuge.
Note: Start timing when the rotor starts moving.
12. To continue, resuspend the samples in injection buffer.
To store, cover with aluminum foil, and store at 4 °C.
Note: Make sure the wells are dry. You may use a Speed-Vac
for 15 min to dry the plate.
IMPORTANT! Make sure the samples are protected from
light while they are drying.
BigDye® Terminator v1.1 Cycle Sequencing Kit
DRAFT
August 27, 2002 9:57 am, 04_PurifyExtension.fm
4-7
Chapter 4
Purifying Extension Products
Precipitating in
384-Well
Reaction Plates
CHEMICAL HAZARD. EDTA. Exposure
causes eye irritation. Read the MSDS, and follow the handling
instructions. Wear appropriate protective eyewear, clothing, and
gloves.
CHEMICAL HAZARD. Ethanol is a
flammable liquid and vapor. Exposure causes eye, skin, and
respiratory tract irritation and may cause central nervous system
depression and liver damage. Read the MSDS, and follow the
handling instructions. Wear appropriate protective eyewear, clothing,
and gloves.
To precipitate 10 µL sequencing reactions in 384-well reaction
plates:
1. Remove the 384-well reaction plate from the thermal cycler
and briefly spin.
2. Add 1 µL of 125 mM EDTA to each well.
Note: Make sure the EDTA reaches the bottom of the wells.
3. Add 1 µL of 3 M sodium acetate to each well.
Note: Make sure the sodium acetate reaches the bottom of
the wells.
4. Add 25 µL of 100% ethanol to each well.
5. Seal the plate with aluminum tape and mix by inverting
4 times.
6. Incubate at room temperature for 15 min.
4-8
DRAFT
BigDye® Terminator v1.1 Cycle Sequencing Kit
August 27, 2002 9:57 am, 04_PurifyExtension.fm
Ethanol/EDTA/Sodium Acetate Precipitation
To precipitate 10 µL sequencing reactions in 384-well reaction
plates: (continued)
7.
If you are using ...
Then ...
a Beckman Allegra
6A centrifuge with a
GH-3.8A rotor
set it at 4 °C and spin the plate at
1650 × g for 45 min.
any other centrifuge
use a plate adapter and spin the plate
at the maximum speed as follows:
• 1400–2000 × g for 45 min
or
• 2000–3000 × g for 30 min
IMPORTANT! Proceed to the next step immediately. If this is
not possible, then spin the plate for an additional 2 min
before performing the next step.
8. Invert the plate and spin up to 185 × g, then remove from the
centrifuge.
9. Add 35 µL of 70% ethanol to each well.
10. With the centrifuge set to 4 °C, spin at 1650 × g for 15 min.
11. Invert the plate and spin up to 185 × g for 1 min, then remove
from the centrifuge.
Note: Start timing when the rotor starts moving.
12. To continue, resuspend the samples in injection buffer.
To store, cover with aluminum foil, and store at 4 °C.
Note: Make sure the wells are dry. You may use a Speed-Vac
for 15 min to dry the plate.
IMPORTANT! Make sure the samples are protected from
light while they are drying.
BigDye® Terminator v1.1 Cycle Sequencing Kit
DRAFT
August 27, 2002 9:57 am, 04_PurifyExtension.fm
4-9
Chapter 4
Purifying Extension Products
Plate and Spin Column Purification
Overview
This section describes the recommended plate and spin columns for
purifying extension products.
IMPORTANT! Extra caution is required when dispensing samples
onto the column bed. Residual dye peaks will result if samples flow
through the sides of the column.
Recommended
Spin Columns
Optimizing Spin
Column
Purification
We recommend Centri-Sep™ spin columns (Applied Biosystems,
PN 401763 for 32 columns and PN 401762 for 100 columns).
IMPORTANT! When using the BigDye terminators v1.1, hydrate the
column for 2 hours (see below).
Tips for optimizing spin column purification when using individual
columns:
• Do not process more columns than you can handle conveniently at
one time.
• Load the sample in the center of the column bed slowly. Make sure
that the sample does not touch the sides of the column and that the
pipet tip does not touch the gel surface.
Note: If samples are not properly loaded, peaks from unincorporated
dye terminators can result.
• Spin the column at 325–730 × g for best results. Use the following
formula to calculate the best speed for your centrifuge:
g = 11.18 × r × (rpm/1000)2
where:
g = relative centrifugal force
r = radius of the rotor in cm
rpm = revolutions per minute
• Do not spin for more than 2 minutes.
• Perform the entire procedure without interruption to ensure
optimal results. Do not allow the column to dry out.
4-10
DRAFT
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August 27, 2002 9:57 am, 04_PurifyExtension.fm
Plate and Spin Column Purification
Performing Spin
Column
Purification
To perform spin column purification:
1. Gently tap the column to cause the gel material to settle to
the bottom of the column.
2. Remove the upper end cap and add 0.8 mL of deionized
water.
3. Replace the upper end cap and vortex or invert the column a
few times to mix the water and gel material.
4. Allow the gel to hydrate at room temperature for at least 2
hours.
Note: Hydrated columns can be stored for a few days at
2–6 °C. Longer storage in water is not recommended. Allow
columns stored at 2–6 °C to warm to room temperature
before use.
5. Remove any air bubbles by inverting or tapping the column
and allowing the gel to settle.
6. Remove the upper end cap first, then remove the bottom cap.
Allow the column to drain completely by gravity.
Note: If flow does not begin immediately, apply gentle
pressure to the column with a pipette bulb.
7. Insert the column into the wash tube provided.
8. Spin the column in a microcentrifuge at 730 × g for
2 minutes to remove the interstitial fluid.
9. Remove the column from the wash tube and insert it into a
sample collection tube (for example, a 1.5-mL
microcentrifuge tube).
10. Remove the extension reaction mixture from its tube and
load it carefully onto the center of the gel material.
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DRAFT
August 27, 2002 9:57 am, 04_PurifyExtension.fm
4-11
Chapter 4
Purifying Extension Products
To perform spin column purification: (continued)
11. Spin the column in a microcentrifuge at 730 × g for
2 minutes.
Note: If using a centrifuge with a fixed-angle rotor, place
the column in the same orientation as it was in for the first
spin. This is important because the surface of the gel will be
at an angle in the column after the first spin.
12. Discard the column. The sample is in the sample collection
tube.
13. Dry the sample in a vacuum centrifuge for 10–15 minutes, or
until dry. Do not over-dry.
4-12
DRAFT
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August 27, 2002 9:57 am, 04_PurifyExtension.fm
Plate Purification Using SDS/Heat Treatment
Plate Purification Using SDS/Heat Treatment
Overview
Preparing
Extension
Products
This section provides instructions for adding a sodium dodecyl
sulfate (SDS)/heat treatment to the spin plate purification method.
This SDS/heat treatment effectively eliminates high concentrations of
unincorporated dye terminators from cycle sequencing reactions.
Use this procedure to prepare extension products for 96-well spin
plate purification.
CHEMICAL HAZARD. Sodium dodecyl
sulfate (SDS). Exposure causes eye, skin, and respiratory tract
irritation. Exposure may cause severe allergic respiratory and skin
reaction. Read the MSDS, and follow the handling instructions. Wear
appropriate protective eyewear, clothing, and gloves.
To prepare extension products:
1. Prepare a solution of 2.2% SDS in deionized water. This
SDS solution is stable at room temperature.
2. Add an appropriate amount of the 2.2% SDS solution to each
sample to bring the final SDS concentration to 0.2%.
For example: Add 2 µL of 2.2% SDS solution to each 20-µL
completed cycle sequencing reaction.
3. Seal the tubes and mix thoroughly.
4. Heat the tubes to 98 °C for 5 min, then allow the tubes to
cool to ambient temperature before proceeding to the next
step.
Note: A convenient way to perform this heating/cooling
cycle is to place the tubes in a thermal cycler and set it as
follows:
• 98 °C for 5 min
• 25 °C for 10 min
5. Spin down the contents of the tubes briefly.
6. Continue with 96-well plate purification.
BigDye® Terminator v1.1 Cycle Sequencing Kit
DRAFT
August 27, 2002 9:57 am, 04_PurifyExtension.fm
4-13
Chapter 4
Purifying Extension Products
Performing 96-Well Spin Plate Purification
Recommended
96-Well Spin
Plates
Purifying with the
96-Well Spin
Plate
For large-scale procedures, you can use 96-well spin plates, such as
the Gel Filtration Kit from Edge Biosystems.
Note: Other spin plate systems may be used to successfully remove
unincorporated dye terminators. However, due to the large number of
variables associated with using spin plate systems, you will need to
optimize the performance of your system in your own laboratory.
To perform purification with the spin plate:
1. Prepare the extension products according to “Preparing
Extension Products” on page 4-13, if desired.
2. Prepare the 96-well spin plate per the manufacturer’s
instructions.
3. Perform the purification per the manufacturer’s instructions.
IMPORTANT! When using the Edge Biosystems gel
filtration kit only, centrifuge at 850 × g for 2 min.
4-14
DRAFT
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August 27, 2002 9:57 am, 04_PurifyExtension.fm
Selecting Sequencing PrimersA
A
Selecting Sequencing Primers
Overview
The choice of sequencing primer sequence, method of primer
synthesis, and approach to primer purification can have a significant
effect on the quality of the sequencing data obtained in dye
terminator cycle sequencing reactions with this kit.
These decisions are particularly important when sequencing is done
on real-time detection systems where signal strength is critical. Some
of the recommendations given here are based on information that is
general knowledge, while others are based on practical experience
gained by Applied Biosystems scientists.
Optimizing Primer
Selection
The following recommendations are provided to help optimize
primer selection:
• Primers should be at least 18 bases long to ensure good
hybridization.
• Avoid runs of an identical nucleotide, especially guanine, where
runs of four or more Gs should be avoided.
• Keep the G-C content in the range 30–80%.
• For cycle sequencing, primers with melting temperatures (Tm)
above 45 °C produce better results than primers with lower Tm.
• For primers with a G-C content less than 50%, it may be necessary
to extend the primer sequence beyond 18 bases to keep the
Tm>45 °C.
• Use of primers longer than 18 bases also minimizes the chance of
having a secondary hybridization site on the target DNA.
• Avoid primers that have secondary structure or that can hybridize
to form dimers.
• Several computer programs for primer selection are available.
They can be useful in identifying potential secondary structure
problems and determining if a secondary hybridization site exists
on the target DNA.
BigDye® Terminator v1.1 Cycle Sequencing Kit
DRAFT
August 15, 2002 11:28 am, AppA_SelectingPrimers.fm
A-1
Appendix A Selecting Sequencing Primers
A-2
DRAFT
BigDye® Terminator v1.1 Cycle Sequencing Kit
August 15, 2002 11:28 am, AppA_SelectingPrimers.fm
Control DNA Sequence
B
B
Control Sequence
Partial Sequence
of pGEM-3Zf(+)
The pGEM-3Zf(+) sequence below is the sequence of the –21 M13
forward primer, followed by the ensuing 1000 bases.
TGTAAAACGACGGCCAGT (–21 M13 primer)
GAATTGTAAT
ACGACTCACT
ATAGGGCGAA
TTCGAGCTCG
40
GTACCCGGGG
ATCCTCTAGA
GTCGACCTGC
AGGCATGCAA
80
GCTTGAGTAT
TCTATAGTGT
CACCTAAATA
GCTTGGCGTA
120
ATCATGGTCA
TAGCTGTTTC
CTGTGTGAAA
TTGTTATCCG
160
CTCACAATTC
CACACAACAT
ACGAGCCGGA
AGCATAAAGT
200
GTAAAGCCTG
GGGTGCCTAA
TGAGTGAGCT
AACTCACATT
240
AATTGCGTTG
CGCTCACTGC
CCGCTTTCCA
GTCGGGAAAC
280
CTGTCGTGCC
AGCTGCATTA
ATGAATCGGC
CAACGCGCGG
320
GGAGAGGCGG
TTTGCGTATT
GGGCGCTCTT
CCGCTTCCTC
360
GCTCACTGAC
TCGCTGCGCT
CGGTCGTTCG
GCTGCGGCGA
400
GCGGTATCAG
CTCACTCAAA
GGCGGTAATA
CGGTTATCCA
440
CAGAATCAGG
GGATAACGCA
GGAAAGAACA
TGTGAGCAAA
480
AGGCCAGCAA
AAGGCCAGGA
ACCGTAAAAA
GGCCGCGTTG
520
CTGGCGTTTT
TCCATAGGCT
CCGCCCCCCT
GACGAGCATC
560
ACAAAAATCG
ACGCTCAAGT
CAGAGGTGGC
GAAACCCGAC
600
AGGACTATAA
AGATACCAGG
CGTTTCCCCC
TGGAAGCTCC
640
BigDye® Terminator v1.1 Cycle Sequencing Kit
DRAFT
August 15, 2002 11:26 am, AppB_ControlSeq.fm
B-1
Appendix B
Control DNA Sequence
CTCGTGCGCT
CTCCTGTTCC
GACCCTGCCG
CTTACCGGAT
680
ACCTGTCCGC
CTTTCTCCCT
TCGGGAAGCG
TGGCGCTTTC
720
TCATAGCTCA
CGCTGTAGGT
ATCTCAGTTC
GGTGTAGGTC
760
GTTCGCTCCA
AGCTGGGCTG
TGTGCACGAA
CCCCCCGTTC
800
AGCCCGACCG
CTGCGCCTTA
TCCGGTAACT
ATCGTCTTGA
840
GTCCAACCCG
GTAAGACACG
ACTTATCGCC
ACTGGCAGCA
880
GCCACTGGTA
ACAGGATTAG
CAGAGCGAGG
TATGTAGGCG
920
GTGCTACAGA
GTTCTTGAAG
TGGTGGCCTA
ACTACGGCTA
960
CACTAGAAGG
ACAGTATTTG
GTATCTGCGC
TCTGCTGAAG
1000
B-2
DRAFT
BigDye® Terminator v1.1 Cycle Sequencing Kit
August 15, 2002 11:26 am, AppB_ControlSeq.fm
Sample Electrophoresis
C
C
Some Important Reminders
• Dye set/primer (mobility) files for the BigDye® terminators v1.1
are the same as those for BigDye terminators original, but different
than those for the dRhodamine terminators and BigDye
terminators v3.1.
• If a mobility file for the wrong sequencing chemistry is used, some
bases may be miscalled due to different dye labeling for the
different chemistries.
Note: See “Required Software” on page 1-4 for information on
obtaining the v1.1 dye set/primer (mobility) files.
BigDye® Terminator v1.1 Cycle Sequencing Kit
DRAFT
August 26, 2002 11:39 am, AppC_Electrophoresis.fm
C-1
Appendix C Sample Electrophoresis
Electrophoresis on the ABI PRISM 3700 DNA
Analyzer
Requirements
Run Modules
Configuration
Run Module
POP-5™ polymer, 50-cm
Seq1_1POP5DefaultModule
Seq1_2POP5DefaultModule
POP-6™ polymer, 50-cm
Seq1_1POP6DefaultModule
Seq1_2POP6DefaultModule
Dye Set/Primer (Mobility) Files
Polymer
Dye Set/Primer (Mobility) File
POP-5 polymer
DT3700POP5{BD}v3.mob
POP-6 polymer
DT3700POP6{BD}v5.mob
Standards
IMPORTANT! Use Dye Set E.
Dye Set
E
Standards
3700 BigDye® Terminator v1.1 Matrix Standard
(PN 4336825)
Note: Refer to the product insert for instructions on using the
standards for this instrument.
C-2
DRAFT
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August 26, 2002 11:39 am, AppC_Electrophoresis.fm
Electrophoresis on the ABI PRISM 3700 DNA Analyzer
Performing
Sample
Electrophoresis
For information on how to perform sample electrophoresis on the
3700 instrument, refer to the following manuals:
• ABI PRISM 3700 DNA Analyzer Sequencing Chemistry Guide
(PN 4309125)
• ABI PRISM 3700 DNA Analyzer User’s Manual (PN 4306152)
BigDye® Terminator v1.1 Cycle Sequencing Kit
DRAFT
August 26, 2002 11:39 am, AppC_Electrophoresis.fm
C-3
Appendix C Sample Electrophoresis
Electrophoresis on the ABI PRISM 3100 and 3100Avant Genetic Analyzers
Requirements
Electrophoresis and data analysis of samples on the ABI PRISM ®
3100 and 3100-Avant Genetic Analyzers require the following:
Run Modules
Configuration
Run Module
POP-4™ polymer, 36-cm
UltraSeq_POP4Default Module
POP-4 polymer, 80-cm
LongSeq80_POP4DefaultModule
POP-6™ polymer, 36-cm
RapidSeq36_POP6DefaultModule
POP-6 polymer, 50-cm
StdSeq50_POP6DefaultModule
Dye Set/Primer (Mobility) Files
Polymer
Dye Set/Primer (Mobility) File
POP-4™ polymer
DT3100POP4LR{BD}v1.mob
POP-6™ polymer
DT3100POP6{BD}v1.mob
Standards
IMPORTANT! Use Dye Set E.
Dye Set
E
Standards
3100 BigDye® Terminator v1.1 Matrix Standard
(PN 4336824)
Note: Refer to the product insert for instructions on using the
standards for this instrument.
C-4
DRAFT
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August 26, 2002 11:39 am, AppC_Electrophoresis.fm
Electrophoresis on the ABI PRISM 3100 and 3100-Avant Genetic Analyzers
Performing
Sample
Electrophoresis
For information on how to perform sample electrophoresis on the
3100 instrument, refer to the following manuals:
• ABI PRISM 3100 Genetic Analyzer Sequencing Chemistry Guide
(PN 4315831)
• ABI PRISM 3100 Genetic Analyzer User’s Manual (PN 4315834)
BigDye® Terminator v1.1 Cycle Sequencing Kit
DRAFT
August 26, 2002 11:39 am, AppC_Electrophoresis.fm
C-5
Appendix C Sample Electrophoresis
Electrophoresis on the ABI PRISM 310 Genetic
Analyzer
Requirements
Electrophoresis and data analysis of samples on the ABI PRISM ® 310
Genetic Analyzer requires the following:
Filter Set E Run Modules
Configuration
Run Module
POP-4™ polymer, 1-mL syringe,
47-cm, 50-µm i.d. capillary, Ld = 36
cm
P4StdSeq (1 mL) E
POP-4 polymer, Rapid Sequencing,
1-mL syringe, 47-cm, 50-µm i.d.
capillary, Ld = 36 cm
P4RapidSeq (1 mL) E
POP-6™ polymer, 1-mL syringe, 61cm, 50-µm i.d. capillary
Seq POP6 (1 mL) E
POP-6 polymer, Rapid Sequencing,
1-mL syringe, 47-cm, 50-µm i.d.
capillary
Seq POP6 Rapid (1 mL) E
Dye Set/Primer (Mobility) Files
Polymer
Operating
System
POP-4™ polymer
Dye Set/Primer (Mobility) File
DT310POP4{BD}v2.mob
NT
®
POP-6™ polymer
POP-4™ polymer
DT310POP6{BD-LR}v3.mob
DT310POP6{BD}.mob
DT310POP4{BD}v2.mob
Macintosh®
POP-6™ polymer
C-6
DRAFT
DT POP6{BD Set-AnyPrimer}
BigDye® Terminator v1.1 Cycle Sequencing Kit
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Electrophoresis on the ABI PRISM 310 Genetic Analyzer
Standards
IMPORTANT! The instrument (matrix) file for the BigDye
terminators v1.1 cannot be used for the BigDye terminators v3.0 or
v3.1.
Dye/Filter
Set
E
Standards for Instrument (Matrix) File Generation
310/377 BigDye Terminator v1.1 Matrix Standards
(PN 4336805)
Note: Refer to the product insert for instructions on using the
standards for this instrument.
Performing
Sample
Electrophoresis
For information on how to perform sample electrophoresis on the
310 instrument, refer to the following manuals:
• ABI PRISM 310 Genetic Analyzer Sequencing Chemistry Guide
(PN 4303189)
• ABI PRISM 310 Genetic Analyzer User’s Manual (PN 4317588)
BigDye® Terminator v1.1 Cycle Sequencing Kit
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C-7
Appendix C Sample Electrophoresis
Electrophoresis on the ABI PRISM 377 DNA
Sequencers
Requirements
Electrophoresis and data analysis of samples on the ABI PRISM ® 377
DNA Sequencers (all models*) require the following:
Filter Set E Run Modules
Configuration*
Run Module
36-cm wtr, 1200 scans/hr, any comb
Seq Run 36E-1200
36-cm wtr, 2400 scans/hr, any comb
Seq Run 36E-2400
48-cm wtr, 1200 scans/hr, any comb
Seq Run 48E-1200
*Any plate check and prerun module can be used on the ABI PRISM 377 DNA
Sequencers.
Dye Set/Primer (Mobility) Files
Gel Formulation
4.5% acrylamide
(29:1) or 5% Long
Ranger™ gel
Operating
System
NT
Macintosh
Dye Set/Primer (Mobility) File
DT377{BD}.mob
DT{BD Set Any-Primer}
*Includes the ABI PRISM 377, ABI PRISM 377-18, ABI PRISM 377 with XL Upgrade, and
the ABI PRISM 377 with 96-Lane Upgrade instruments.
C-8
DRAFT
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Electrophoresis on the ABI PRISM 377 DNA Sequencers
Standards
IMPORTANT! The instrument (matrix) file for the BigDye
terminators v1.1 cannot be used for the BigDye terminators v3.0 or
v3.1.
Dye/Filter
Set
E
Standards for Instrument (Matrix) File Generation
310/377 BigDye Terminator v1.1 Matrix Standards
(PN 4336805)
Note: Refer to the product insert for instructions on using the
standards for this instrument.
Using the
Lane Guide Kit
If you are using the BigDye® chemistries v1.1 on the 377 instrument
in conjunction with the ABI PRISM ® Lane Guide™ Lane
Identification Kit, refer to that kit’s protocol (PN 4313804) for
instructions on resuspending and loading samples.
Using Long-Read
Gel and Buffer
Formulations
For longer sequencing read lengths follow the gel and buffer
formulations described in the user bulletin entitled Achieving Longer
High Accuracy Reads on the 377 Sequencer (PN 4315153).
Performing
Sample
Electrophoresis
For information on how to perform sample electrophoresis on the
377 instrument, refer to the following manuals:
• Automated DNA Sequencing Chemistry Guide (PN 4305080)
• ABI PRISM 377 DNA Sequencer User’s Manual (PN 4307164)
BigDye® Terminator v1.1 Cycle Sequencing Kit
DRAFT
August 26, 2002 11:39 am, AppC_Electrophoresis.fm
C-9
Appendix C Sample Electrophoresis
C-10
DRAFT
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Troubleshooting
D
D
Observation
Possible Causes
Recommended Actions
No recognizable
sequence
Insufficient template
• Quantitate DNA template
• Increase amount of DNA in the
sequencing reactions
Inhibitory contaminant in
the template
Clean up the template
Insufficient primer
• Quantitate the primer
• Increase amount of primer in the
sequencing reactions
Primer has no annealing
site
Use a primer that is complementary to the
template
Poor primer design or
incorrect primer sequence
Redesign the primer
Missing reagent
Repeat the reactions, carefully following
the protocol
Old or mishandled
reagents
Use fresh reagents
Thermal cycler power
failure
Repeat the reactions
Thermal cycling
conditions incorrect
•
•
•
•
Extension products lost
during reaction cleanup
• Make sure you use the correct
centrifugation speeds and times for
precipitation and spin column
procedures
• Make sure the ethanol concentration is
correct for the precipitation protocols
BigDye® Terminator v1.1 Cycle Sequencing Kit
Calibrate the thermal cycler regularly
Use correct thermal cycling parameters
Use correct tube for your thermal cycler
Set ramp rate to 1 °C/sec
DRAFT
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D-1
Observation
Possible Causes
Recommended Actions
No recognizable
sequence (continued)
Extension products not
resuspended
Carefully resuspend the sample pellet in
loading buffer
Lane tracking failure (377
instrument)
Check the lane tracking. If necessary,
retrack and reextract the lanes
Electrokinetic injection
failure (capillary
instruments)
Repeat the injections
Insufficient DNA in the
sequencing reactions
• Use more DNA in the sequencing
reactions
• Load or inject more of the resuspended
sequencing reactions
Degraded template
Prepare fresh DNA and repeat the
reactions
Old or mishandled
reagents
Use fresh reagents
Thermal cycling
conditions incorrect
•
•
•
•
Lane tracking failure (377
instrument)
Check the lane tracking. If necessary,
retrack and reextract the lanes
Electrokinetic injection
failure (capillary
instruments)
Repeat the injections
Inhibitory contaminant in
the template
Clean up the template
Multiple templates in the
sequencing reaction
Examine template on an agarose gel to be
sure only one template is present.
Multiple priming sites
• Make sure primer has only one priming
site.
• If necessary, redesign primer
Multiple primers when
sequencing PCR products
Purify your PCR template to remove
excess primers
Noisy data throughout the
sequence with low signal
strength
Noisy data throughout the
sequence with good
signal strength
Calibrate the thermal cycler regularly
Use correct thermal cycling parameters
Use correct tube for your thermal cycler
Set ramp rate to 1 °C/sec
Observation
Possible Causes
Recommended Actions
Noisy data throughout the
sequence with good
signal strength (continued)
Primer with N-1
contamination
Use HPLC-purified primer
High signal saturating the
detector
• Use less DNA in the sequencing
reactions
• Load or inject less of the resuspended
sequencing reactions
Incorrect run module
Use correct run module
Incorrect instrument
(matrix) file
Use correct instrument file for terminator
chemistry
Mixed plasmid separation
Make sure you have only one template
Multiple PCR products
Make sure you have only one template
Primer-dimer
contamination in PCR
sequencing
• Optimize your PCR amplification
• Make sure there is no sequence
complementarity between the two PCR
primers
• Make sure your sequencing primer
does not overlap the sequences of the
PCR primers
• Use a Hot Start technique such as with
Amplitaq® Gold® polymerase
Slippage after repeat
region in template
• Try an alternate sequencing chemistry
• Use an anchored primer
Incorrect dye set/primer
(mobility) file
Use correct dye set/primer file
Incorrect Peak 1 location
for data analysis
Select a new Peak 1 location
Gel with very different
separation properties than
the gel matrices used to
construct the dye
set/primer (mobility) file
Use correct dye set/primer file for your gel
type
Noise up to or after a
specific point in the
sequence
Poor mobility correction
BigDye® Terminator v1.1 Cycle Sequencing Kit
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D-3
Appendix D
Troubleshooting
Observation
Possible Causes
Recommended Actions
Excess dye peaks at
beginning of sequence
Poor removal of
unincorporated dye
terminators
Use ethanol/EDTA precipitation protocol
to remove unincorporated dye terminators
With Centri-Sep spin columns, take care
to load sample on the center of the gel
surface
Note: Do not touch the gel surface with
the pipet tip.
IMPORTANT! Be sure you hydrate the
column for at least 2 hours.
Spin samples for recommended times
(spinning too long precipitates more dyes
with the sample)
With microfuge tubes, aspirate the
supernatant rather than decanting
(decanting leaves excess ethanol on the
sides of the tube)
Select the start point for data analysis to
exclude excess dye peaks
Pull-up peaks or
bleedthrough
Total signal strength over
4000
• Quantitate DNA template
• Decrease amount of DNA in the
sequencing reactions
Load or inject less of the resuspended
sequencing reactions
D-4
DRAFT
BigDye® Terminator v1.1 Cycle Sequencing Kit
August 27, 2002 10:01 am, AppD_Troubleshooting.fm
Obtaining Technical Support E
E
Services and Support
Applied
Biosystems
Web Site
A services and support page is available on the Applied Biosystems
Web site. To access this, go to:
http://www.appliedbiosystems.com
and click the link for services and support.
At the services and 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 services and support page provides worldwide
telephone and fax numbers to contact Applied Biosystems Technical
Support and Sales facilities.
BigDye® Terminator v1.1 Cycle Sequencing Kit
DRAFT
August 20, 2002 2:48 pm, AppE_TechSupport.fm
E-1
Appendix E
E-2
Obtaining Technical Support
DRAFT
BigDye® Terminator v1.1 Cycle Sequencing Kit
August 20, 2002 2:48 pm, AppE_TechSupport.fm
Index
Numerics
310 requirements
mobility files C-6
run modules C-6
sequencing standards C-7
3100 requirements
mobility files C-4
run modules C-4
spectral calibration standards
3700 requirements
mobility files C-2
run modules C-2
spectral calibration standards
377 requirements
matrix file standards C-9
mobility files C-8
run modules C-8
cycle sequencing
BAC DNA 3-7
protocol for GeneAmp
D
disposables
cycle sequencing 1-8
purifying 1-9
DNA
quantity characteristics 2-6
quantity to use 2-6
dye set/primer files, downloading
dye/filter set
3100 1-4
3700 1-4
377 1-4
C-4
C-2
1-6
E
A
attention words
3-5
ethanol/EDTA precipitation
384-well 4-4
96-well 4-3
ethanol/EDTA/sodium acetate
precipitation in 384-well 4-8
precipitation in 96-well 4-6
1-11
B
bacterial artificial chromosomes
2-3
C
centrifuge, recommended 1-9
components, sequencing kits 1-6
contaminants, of templates 2-5
control
double-stranded 2-1
electrophoresis 2-1
single-stranded 2-1
control DNA, reason for 2-1
customer support. See technical support
F
formulation, new 1-1
freeze-thaw, limits 1-7
H
hazards
chemical 1-11
waste 1-12
E-1
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Index-1
I
R
instruments
sequencing 1-3
thermal cyclers 1-3
reaction mixtures
BAC DNA 3-7
for 384-well 3-4
for 96-well 3-2
for microcentrifuge tubes
reagents
electrophoresis 1-10
purifying 1-9
required files
matrix 1-1
spectral 1-1
L
Lane Guide C-9
long read lengths C-9
M
matrix standards
310 1-4
377 1-4
general requirements
mobility files 1-2
about C-1
downloading 1-6
MSDSs, ordering 1-13
S
1-2
P
pGEM-3Zf(+) sequence B-1
primers
importance of selection A-1
optimizing selection A-1
purification
SDS and 96-well 4-13
spin plate 4-14
purifying
methods 2-3, 4-1
methods for PCR 2-3
PCR by ultrafiltration 2-4
Q
quality problems, template 2-5
quality, examining DNA 2-5
quantitating DNA 2-6
Index-2
3-2
DRAFT
safety
chemical 1-11 to 1-13
guide 1-11 to 1-13
SDS, and purification 4-13
sequencing buffer
optimizing 1-2
sequencing kits, ordering 1-6
sequencing problems 2-4
sequencing standards
for each instrument 2-2
general requirements 1-2
shorter read lengths, and DNA quantity 2-6
signal intensities, and DNA quantity 2-6
site preparation and safety guide 1-11
spectral standards
3100 1-4
3700 1-4
spin column purification
optimizing 4-10
performing 4-11
storage temperatures 1-7
BigDye® Terminator v1.1 Cycle Sequencing Kit
August 27, 2002 10:02 am, 4337036A_v1.1IX.fm
T
technical support E-1
templates
BAC DNA 2-3
PCR 2-3
poor quality characteristics 2-4
preparing 2-3
top-heavy data, and DNA quantity 2-6
U
user attention words
1-11
W
warning symbols
1-11
BigDye® Terminator v1.1 Cycle Sequencing Kit
DRAFT
August 27, 2002 10:02 am, 4337036A_v1.1IX.fm
Index-3
Index-4
DRAFT
BigDye® Terminator v1.1 Cycle Sequencing Kit
August 27, 2002 10:02 am, 4337036A_v1.1IX.fm
Headquarters
850 Lincoln Centre Drive
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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.
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 USA, 08/2010
Part Number 4337036 Rev. B
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