DRAFT
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BigDye
Terminator v3.1
Cycle Sequencing Kit
Protocol
DRAFT
August 27, 2002 12:32 pm, 4337035A_v3.1Title.fm
 Copyright 2002, 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 5,332,666 and 5,821,058 or their foreign counterpart claims, has an up-front fee
component and a running-royalty component. The purchase price of BigDye
Terminator v3.1 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 v3.1 Cycle Sequencing Kit includes a limited, nontransferable, non-exclusive license
(without the right to resell, repackage, or sublicense) under the process claims of one or more of U.S. Patents 5,800,996, 5,863,727,
and 5,945,526, 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; 4,994,372, U.S. Patent Application Serial Nos. 08/324437; 08/337615 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
Applera Corporation 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 Applera Corporation 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 4337035 Rev. A
09/2002
DRAFT
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BigDye
Terminator v3.1 Cycle Sequencing Kit iii
Contents
Chapter 1 Introduction
Chapter Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1
In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1
About the Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1
New Formulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1
Features and Compatibilities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1
BigDye Terminator v3.1 Cycle Sequencing Kit . . . . . . . . . . . . . . . 1-2
Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3
Instrument Platforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3
Thermal Cyclers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3
Required Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4
Dye/Filter Sets and Matrix Standards for the 310
and 377 Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4
Dye Sets and Spectral Standards for the 3700, 3100,
and 3100-Avant Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-5
Instructions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-5
Dye Set/Primer (Mobility) Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-6
Reagents and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-6
Available Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-6
Description of Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-6
Storage and Use of the Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-7
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
DRAFT
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iv BigDye
Terminator v3.1 Cycle Sequencing Kit
Chapter 2 Preparing the Templates
Chapter Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1
In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1
Control DNA Templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1
Using Control DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1
Control DNA Sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1
An Additional Control Sold Separately . . . . . . . . . . . . . . . . . . . . . 2-1
Template Preparation Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-3
Single- and Double-Stranded Templates . . . . . . . . . . . . . . . . . . . 2-3
BAC DNA Templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-3
PCR Templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-3
Purifying PCR Fragments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-4
DNA Quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-4
Poor Template Quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-4
Contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-4
Determining DNA Quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-1
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-1
Cycle Sequencing Single- and Double-Stranded DNA . . . . . . . . . . . . 3-2
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-2
Preparing the Reactions for 96-Well Reaction Plates
or Microcentrifuge Tubes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-2
Using BigDye Terminator v1.1/3.1 Sequencing Buffer . . . . . . . . . 3-3
Preparing the Reactions for 384-Well Plates . . . . . . . . . . . . . . . . . 3-4
Cycle Sequencing on the System 9700, 9600, 2700, or 2400 . . . 3-5
Cycle Sequencing Large DNA Templates . . . . . . . . . . . . . . . . . . . . . . 3-5
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-5
Thermal Cyclers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-5
Preparing Sequencing Reactions . . . . . . . . . . . . . . . . . . . . . . . . . 3-6
Performing Cycle Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-6
DRAFT
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BigDye
Terminator v3.1 Cycle Sequencing Kit v
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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-2
Recommended Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-2
Precipitating in 96-Well Reaction Plates . . . . . . . . . . . . . . . . . . . . 4-3
Precipitating in 384-Well Reaction Plates . . . . . . . . . . . . . . . . . . . 4-5
Ethanol/EDTA/Sodium Acetate Precipitation . . . . . . . . . . . . . . . . . . . . 4-7
Precipitating in 96-Well Reaction Plates . . . . . . . . . . . . . . . . . . . . 4-7
Precipitating in 384-Well Reaction Plates . . . . . . . . . . . . . . . . . . . 4-9
Plate and Spin Column Purification . . . . . . . . . . . . . . . . . . . . . . . . . . 4-11
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-11
Preparing Extension Products . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-11
Performing Spin Column Purification . . . . . . . . . . . . . . . . . . . . . . 4-12
Recommended Spin Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-12
Optimizing Spin Column Purification . . . . . . . . . . . . . . . . . . . . . . 4-12
Preparing the Spin Column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-13
Purifying with the Spin Column . . . . . . . . . . . . . . . . . . . . . . . . . . 4-14
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
DRAFT
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vi BigDye
Terminator v3.1 Cycle Sequencing Kit
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 . . . . . . . . . . . C-8
Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-8
Using the Lane Guide Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-9
Using Long-Read Gel and Buffer Formulations . . . . . . . . . . . . . . . C-9
Performing Sample Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . C-9
Appendix D Troubleshooting
Appendix E Obtaining Technical Support
Services and Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-1
Applied Biosystems Web Site . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-1
Index
DRAFT
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BigDye
Terminator v3.1 Cycle Sequencing Kit 1-1
1Introduction 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 v3.1 Cycle Sequencing Kit has a new
formulation that delivers:
* Increased robustness
* More even peak heights
* Longer read lengths
The thermal cycling and clean up protocols for cycle sequencing have
been modified to optimize results using the new formulation.
Features and
Compatibilities
* The BigDye Terminator v3.1 Cycle Sequencing Kit 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 theABI PRISM
3700 DNA Analyzer,
ABI PRISM
3100 Genetic Analyzer, and ABI PRISM
3100-Avant
Genetic Analyzer.
DRAFT
August 27, 2002 12:26 pm, 01_Introduction.fm
Chapter 1 Introduction
1-2 BigDye
Terminator v3.1 Cycle Sequencing Kit
Applied Biosystems recommends that you verify the quality of
your current matrix or spectral before proceeding. If it is necessary
to generate a new matrix or spectral, use the appropriate matrix
and/or sequencing standard for your instrument.
­ The 310 and 377 instruments use the 310/377 BigDye
Terminator v3.1 Matrix Standards (PN 4336948) for instrument
(matrix) file generation.
­ The 3700 instrument requires the 3700 BigDye
Terminator
v3.1 Matrix Standard (PN 4336975) for spectral calibration.
­ The 3100 and 3100-Avant instruments require the 3100
BigDye
Terminator v3.1 Matrix Standard (PN 4336974) for
spectral calibration.
* The alcohol precipitation methods are different from those
recommended for earlier versions of BigDye
terminators.
* The existing mobility files can be used with their respective
platforms. New mobility files are not necessary.
* The basecallers are the same.
BigDye
Terminator v3.1
Cycle
Sequencing Kit
The BigDye Terminator v3.1 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
the 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.
DRAFT
August 27, 2002 12:26 pm, 01_Introduction.fm
Instruments
BigDye
Terminator v3.1 Cycle Sequencing Kit 1-3
Instruments
Instrument
Platforms
The BigDye Terminator v3.1 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.
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°/second), 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.
DRAFT
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Chapter 1 Introduction
1-4 BigDye
Terminator v3.1 Cycle Sequencing Kit
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.
IMPORTANT! The instrument (matrix) file for the BigDye
terminators v3.1 cannot be used for the BigDye terminators
(original), BigDye terminators v2.0, dRhodamine terminators, or
BigDye
primers (original), but can be used for BigDye terminators
v3.0.
Instrument Dye/Filter Set
Standards for Instrument
(Matrix) File Generation
310 Genetic Analyzer Filter Set E 310/377 BigDye
Terminator v3.1 Matrix
Standards (PN 4336948)*
*The BigDye Terminator v3.1 Cycle Sequencing Kit does not require a new
spectral calibration file if you currently have a BigDye Terminator v3.0
spectral file on your instrument.
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.
Filter Set E
DRAFT
August 27, 2002 12:26 pm, 01_Introduction.fm
Required Software
BigDye
Terminator v3.1 Cycle Sequencing Kit 1-5
Dye Sets and
Spectral
Standards for the
3700, 3100, and
3100-Avant
Instruments
IMPORTANT! Spectral calibrations for the BigDye terminators v3.1
are not compatible with the BigDye terminators (original), BigDye
terminators v2.0, dRhodamine terminators, or BigDye primers
(original).
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 v3.1 to generate matrices.
For Performing Spectral Calibrations
* For the 3700 instrument, refer to the product insert for instructions
on using the 3700 BigDye Terminator v3.1 Matrix or Sequencing
Standard to perform spectral calibration.
* For the 3100 and 3100-Avant instruments, refer to the product
insert for instructions on using the BigDye Terminator v3.1 Matrix
or Sequencing Standard to perform spectral calibration.
Instrument Dye Set
Standards for Spectral
Calibration
3700 DNA Analyzer
with Data Collection
v2.0
3700 DNA Analyzer
with Data Collection
v1.1 and v1.1.1
H
D
3700 BigDye
Terminator v3.1
Matrix Standard (PN 4336975)*
*The BigDye Terminator v3.1 Cycle Sequencing Kit does not require a new
spectral calibration file if you currently have a BigDye
Terminator v3.0
spectral file on your instrument.
3100 Genetic Analyzer
3100-Avant Genetic
Analyzer
Z 3100 BigDye
Terminator v3.1
Matrix Standard (PN 4336974)*
DRAFT
August 27, 2002 12:26 pm, 01_Introduction.fm
Chapter 1 Introduction
1-6 BigDye
Terminator v3.1 Cycle Sequencing Kit
Dye Set/Primer
(Mobility) Files
To analyze sequencing data generated with BigDye
chemistries
v3.1, you need dye set/primer (mobility) files that were created for
v3.0 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 BigDye
Terminator v3.1 Cycle Sequencing Kit Protocol
(PN 4337035) is available separately and can be ordered at no charge.
Description of
Reagents
A listing of the kit reagents is given below.
* Ready Reaction Mix
* pGEM-3Zf(+) double-stranded DNA Control Template
* ­21 M13 Control Primer (forward)
* BigDye Terminator v1.1/3.1 Sequencing Buffer (5X)
The following kits are available:
Kit
Number of
Reactions
Part Number
The BigDye Terminator v3.1 Cycle
Sequencing Kit*
*The BigDye Terminator v3.1 Cycle Sequencing Kit does not require a new
spectral calibration file if you currently have a BigDye Terminator v3.0
spectral file on your instrument.
100 4337455
1000 4337456
5000 4337457
25000 4337958
DRAFT
August 27, 2002 12:26 pm, 01_Introduction.fm
Materials Supplied by the User
BigDye
Terminator v3.1 Cycle Sequencing Kit 1-7
Storage and Use
of the Kit
* 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 5­10) 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.
DRAFT
August 27, 2002 12:26 pm, 01_Introduction.fm
Chapter 1 Introduction
1-8 BigDye
Terminator v3.1 Cycle Sequencing Kit
Materials for
Cycle
Sequencing
The table below lists the plates or tubes required for the
recommended Applied Biosystems thermal cyclers (page 1-3).
Thermal Cycler Plate or Tube
Applied Biosystems
Part Number
GeneAmp PCR System
9700
MicroAmp 96-Well Reaction Plate N801-0560
384-Well Reaction Plate 4305505
MicroAmp Reaction Tubes, 0.2-mL N801-0533
MicroAmp Caps, 12 or 8/strip N801-0534 or N801-
0535
ABI PRISM
Optical Adhesive Cover
Starter Pack or ABI PRISM
Optical
Adhesive Covers
4313663 or 4311971
GeneAmp PCR System
9600
MicroAmp 96-Well Reaction Plate N801-0560
MicroAmp Reaction Tubes, 0.2-mL N801-0533
MicroAmp Caps, 12 or 8/strip N801-0534 N801-0535
ABI PRISMTM
Optical Adhesive Cover
Starter Pack or ABI PRISM
Optical
Adhesive Covers
4313663 or 4311971
GeneAmp PCR System
2400 and 2700
MicroAmp Reaction Tubes, 0.2-mL N801-0533
MicroAmp Caps, 12 or 8/strip N801-0534 N801-0535
DRAFT
August 27, 2002 12:26 pm, 01_Introduction.fm
Materials Supplied by the User
BigDye
Terminator v3.1 Cycle Sequencing Kit 1-9
Materials for
Purifying
Extension
Products
Other Equipment 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.
Method Material Supplier
Ethanol/EDTA
Precipitation
Ethanol (EtOH), 200
proof, Molecular
Biology grade
EDTA, 125 mM
Sealing tape
MLS
MLS
Costar 6570 Thermowell
Sealing Tape
Ethanol/EDTA/
Sodium Acetate
Precipitation
Ethanol (EtOH), 200
proof, Molecular
Biology grade
Sodium acetate
(NaOAc), 3 M, pH 5.2
Sealing tape
MLS
Applied Biosystems
(PN 400320)
Costar 6570 Thermowell
Sealing Tape
Plate Column
Purification
Note: For 96well
reaction
plates
96-Well columns for
purification
Sealing tape
2.2% SDS in deionized
water
See "Recommended 96Well
Spin Plates" on
page 4-14
Costar 6570 Thermowell
Sealing Tape
See "Plate and Spin
Column Purification" on
page 4-11
Spin Column
Purification
Centri-SepTM spin
column, 1-mL, 32
columns, 100 columns
Sealing tape
2.2% SDS in deionized
water
Applied Biosystems
PN 401763, PN 401762
Costar 6570 Thermowell
Sealing Tape
See "Plate and Spin
Column Purification" on
page 4-11
DRAFT
August 27, 2002 12:26 pm, 01_Introduction.fm
Chapter 1 Introduction
1-10 BigDye
Terminator v3.1 Cycle Sequencing Kit
Materials for
Electrophoresis Instrument Material Supplier
ABI PRISM 3700
DNA Analyzer,
3100 and 3100Avant
Genetic
Analyzers
Hi-DiTM Formamide, 25-mL
bottle
Applied Biosystems
(PN 4311320)
3700/3730 BigDye
Terminator v3.1
Sequencing Standard
BigDye
Terminator v3.1
Sequencing Standard
Applied Biosystems
(PN 4336943)
Applied Biosystems
(PN 4336935)
3700 BigDye Terminator
v3.1 Matrix Standard
3100 BigDye Terminator
v3.1 Matrix Standard
Applied Biosystems
(PN 4336975)
Applied Biosystems
(PN 4336974)
ABI PRISM 310
Genetic Analyzer
Template Suppression
Reagent (TSR)
Applied Biosystems
(PN 401674)
EDTA MLS
BigDye
Terminator v3.1
Sequencing Standard
Applied Biosystems
(PN 4336935)
310/377 BigDye
Terminator v3.1 Matrix
Standards
Applied Biosystems
(PN 4336948)
ABI PRISM 377 DNA
Sequencer
Formamide MLS
EDTA MLS
25 mM EDTA with
50 mg/mL blue dextran
Applied Biosystems
(PN 402055)
BigDye
Terminator v3.1
Sequencing Standard
Applied Biosystems
(PN 4336935)
310/377 BigDye
Terminator v3.1 Matrix
Standards
Applied Biosystems
(PN 4336948)
DRAFT
August 27, 2002 12:26 pm, 01_Introduction.fm
General Safety
BigDye
Terminator v3.1 Cycle Sequencing Kit 1-11
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.
DRAFT
August 27, 2002 12:26 pm, 01_Introduction.fm
Chapter 1 Introduction
1-12 BigDye
Terminator v3.1 Cycle Sequencing Kit
* 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.
DRAFT
August 27, 2002 12:26 pm, 01_Introduction.fm
Chemical Safety
BigDye
Terminator v3.1 Cycle Sequencing Kit 1-13
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:
To obtain documents through the Applied Biosystems Web site:
For chemicals not manufactured or distributed by
Applied Biosystems, call the chemical manufacturer.
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.
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.
DRAFT
August 27, 2002 12:26 pm, 01_Introduction.fm
Chapter 1 Introduction
1-14 BigDye
Terminator v3.1 Cycle Sequencing Kit
DRAFT
August 21, 2002 8:27 am, 02_PrepTemplates.fm
BigDye
Terminator v3.1 Cycle Sequencing Kit 2-1
2Preparing the Templates 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
We recommend 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 B,
"Control DNA Sequence."
An Additional
Control Sold
Separately
The BigDye
Terminator v3.1 Sequencing Standard Kit provides an
additional control to help in troubleshooting electrophoresis runs. It
contains lyophilized sequencing reactions that require only
resuspension and denaturation before use.
DRAFT
August 21, 2002 8:27 am, 02_PrepTemplates.fm
Chapter 2 Preparing the Templates
2-2 BigDye
Terminator v3.1 Cycle Sequencing Kit
There are two v3.1 sequencing standard kits, 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 v3.1
Sequencing Standard
4336943
ABI PRISM
3100 Genetic Analyzer
BigDye
Terminator
v3.1 Sequencing
Standard
4336935
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.
DRAFT
August 21, 2002 8:27 am, 02_PrepTemplates.fm
Template Preparation Methods
BigDye
Terminator v3.1 Cycle Sequencing Kit 2-3
Template Preparation Methods
Single- and
Double-Stranded
Templates
Refer to the Automated DNA Sequencing Chemistry Guide
(PN 4305080) for information on preparing single- and doublestranded
templates.
BAC DNA
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 optimal 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
(PN 4305080) for information on sequencing PCR templates.
*Marra, M., Weinstock, L.A., and Mardis, E.R. 1996. End sequence
determination from large insert cloning using energy transfer fluorescent
primers. Genomic Methods 6: 1118­1122.
DRAFT
August 21, 2002 8:27 am, 02_PrepTemplates.fm
Chapter 2 Preparing the Templates
2-4 BigDye
Terminator v3.1 Cycle Sequencing Kit
Purifying PCR
Fragments
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.
Contamination Potential contaminants include:
* Proteins
* RNA
* Chromosomal DNA
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 the sample. This should yield approximately 40­60
L of sample.
7. Add deionized water to bring the purified PCR fragments to
the original volume.
DRAFT
August 21, 2002 8:27 am, 02_PrepTemplates.fm
DNA Quality
BigDye
Terminator v3.1 Cycle Sequencing Kit 2-5
* 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.
DRAFT
August 21, 2002 8:27 am, 02_PrepTemplates.fm
Chapter 2 Preparing the Templates
2-6 BigDye
Terminator v3.1 Cycle Sequencing Kit
DNA Quantity
Quantitating DNA If possible, quantitate the amount of purified DNA by measuring the
absorbance at 260 nm or by some other method.
Template
Quantity
The table below shows the amount of template to use in a cycle
sequencing reaction.
Note: In general, higher DNA quantities give higher signal
intensities.
The template quantities stated 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.
Template Quantity
PCR product:
100­200 bp
200­500 bp
500­1000 bp
1000­2000 bp
>2000 bp
1­3 ng
3­10 ng
5­20 ng
10­40 ng
20­50 ng
Single-stranded 25­50 ng
Double-stranded 150­300 ng
Cosmid, BAC 0.5­1.0 g
Bacterial genomic DNA 2­3 g
DRAFT
August 27, 2002 9:59 am, 03_PerformSeq.fm
BigDye
Terminator v3.1 Cycle Sequencing Kit 3-1
3Performing Cycle Sequencing 3
Chapter Summary
In This Chapter The following topics are covered in this chapter:
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-1
Cycle Sequencing Single- and Double-Stranded DNA. . . . . . . . . .3-2
Cycle Sequencing Large DNA Templates. . . . . . . . . . . . . . . . . . . .3-5
Introduction
Overview The cycle sequencing protocols used for the BigDye
Terminator
v3.1 Cycle Sequencing Kit have been modified to optimize results
using the new chemistries. Applied Biosystems does not recommend
the use of the protocols for the BigDye
Terminator v1.0 or v2.0
Ready Reaction Cycle Sequencing Kits.
DRAFT
August 27, 2002 9:59 am, 03_PerformSeq.fm
Chapter 3 Performing Cycle Sequencing
3-2 BigDye
Terminator v3.1 Cycle Sequencing Kit
Cycle Sequencing Single- and Double-Stranded
DNA
Overview This section describes how to prepare reactions and perform cycle
sequencing on a variety of templates, including M13, plasmids, and
PCR products.
Preparing the
Reactions for 96Well
Reaction
Plates or
Microcentrifuge
Tubes
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*
*See "Using BigDye Terminator v1.1/3.1 Sequencing Buffer" below.
8.0 L
Template See the table in "Template
Quantity" on page 2-6.
Primer 3.2 pmol
Deionized water q.s.
Total Volume 20 L
2. Mix well and spin briefly.
DRAFT
August 27, 2002 9:59 am, 03_PerformSeq.fm
Cycle Sequencing Single- and Double-Stranded DNA
BigDye
Terminator v3.1 Cycle Sequencing Kit 3-3
Using BigDye
Terminator
v1.1/3.1
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 L final volume, you would use
4 L of ready reaction premix and 2 L of BigDye sequencing buffer
as shown below.
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.
Reagent Concentration Volume
Ready Reaction Premix 2.5X 4 L
BigDye Sequencing
Buffer
5X 2 L
Primer -- 3.2 pmol
Template -- See
"Template
Quantity" on
page 2-6.
Water -- to 20 L
Final Volume 1X 20 L
DRAFT
August 27, 2002 9:59 am, 03_PerformSeq.fm
Chapter 3 Performing Cycle Sequencing
3-4 BigDye
Terminator v3.1 Cycle Sequencing Kit
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 L. Therefore, we recommend doing a 10 L 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*
*Note: For instructions on using BigDye sequencing buffer, see "Using
BigDye Terminator v1.1/3.1 Sequencing Buffer" on page 3-3.
4.0 L
Template See the table in "Template
Quantity" on page 2-6.
Primer 3.2 pmol
Deionized water q.s.
Total Volume 10 L
2. Mix well and spin briefly.
3. Use on a GeneAmp PCR System 9700 Dual 384-Well
Sample Block Module.
DRAFT
August 27, 2002 9:59 am, 03_PerformSeq.fm
Cycle Sequencing Large DNA Templates
BigDye
Terminator v3.1 Cycle Sequencing Kit 3-5
Cycle
Sequencing on
the System 9700,
9600, 2700, or
2400
Cycle 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)
To sequence single- and double-stranded DNA on the GeneAmp
PCR System 9700 (in 9600 emulation mode), 9600, 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 sec
* Rapid thermal ramp to 50 °C
* 50 °C for 5 sec
* Rapid thermal ramp to 60 °C
* 60 °C for 4 min
*Rapid thermal ramp is 1 °C/second.
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."
DRAFT
August 27, 2002 9:59 am, 03_PerformSeq.fm
Chapter 3 Performing Cycle Sequencing
3-6 BigDye
Terminator v3.1 Cycle Sequencing Kit
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 for Cycle Sequencing" on page 1-8.
Performing Cycle
Sequencing
To prepare the sequencing reaction:
1. For each reaction add the following reagents to a separate
tube:
Reagent Quantity
Terminator Ready Reaction
Mix*
*Note: For instructions on using BigDye sequencing buffer, see "Using
BigDye Terminator v1.1/3.1 Sequencing Buffer" on page 3-3.
8.0 L
Template See the table in "Template
Quantity" on page 2-6.
Primer 3.2 pmol
Deionized water q.s.
Total Volume 20 L
2. Mix well and spin briefly.
3. Use on a GeneAmp PCR System 9600 or 9700 Sample
Block Module.
To perform cycle sequencing on BAC DNA:
1. Place the tubes in a thermal cycler and set the volume to
20 L.
2. Heat the tubes at 95 °C for 5 minutes.
DRAFT
August 27, 2002 9:59 am, 03_PerformSeq.fm
Cycle Sequencing Large DNA Templates
BigDye
Terminator v3.1 Cycle Sequencing Kit 3-7
3. Repeat the following for 50 cycles:*
* Rapid thermal ramp
to 95 °C
* 95 °C for 30 sec
* Rapid thermal ramp to 50­55 °C (depending on template)
* 50­55 °C for 10 sec
* Rapid thermal ramp to 60 °C
* 60 °C for 4 min
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.
To perform cycle sequencing on BAC DNA: (continued)
DRAFT
August 27, 2002 9:59 am, 03_PerformSeq.fm
Chapter 3 Performing Cycle Sequencing
3-8 BigDye
Terminator v3.1 Cycle Sequencing Kit
DRAFT
August 27, 2002 12:28 pm, 04_Purifying.fm
BigDye
Terminator v3.1 Cycle Sequencing Kit 4-1
4Purifying Extension Products 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. . . . . . . . . . . . . . . . .4-7
Plate and Spin Column Purification . . . . . . . . . . . . . . . . . . . . . . .4-11
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 components of the BigDye
Terminator v3.1 Cycle Sequencing
Kit have been optimized to produce excellent results under a wide
variety of conditions. However, this kit may require changes to the
clean-up protocols used for previous kits. To obtain clean sequencing
data, Applied Biosystems recommends the following purification
methods:
* Ethanol/EDTA precipitation
* Ethanol/EDTA/sodium acetate precipitation
* Plate and spin column purification
Use the method that works best for your particular application.
DRAFT
August 27, 2002 12:28 pm, 04_Purifying.fm
Chapter 4 Purifying Extension Products
4-2 BigDye
Terminator v3.1 Cycle Sequencing Kit
Note: The precipitation protocols given here have been optimized for
use with the v3.1 formulation at the specified sequencing volumes
(20 L in 96-well format and 10-L 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 v3.1, the ethanol/EDTA precipitation
method produces consistent signal, while minimizing unincorporated
dyes. It is particularly good at getting rid of unincorporated dyelabeled
terminators.
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. This can lead 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
(67­71%).
DRAFT
August 27, 2002 12:28 pm, 04_Purifying.fm
Ethanol/EDTA Precipitation
BigDye
Terminator v3.1 Cycle Sequencing Kit 4-3
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-L sequencing reactions in 96-well reaction
plates:
1. Remove the 96-well reaction plate from the thermal cycler
and briefly spin.
2. Add 5 L of 125 mM EDTA to each well.
Note: Make sure the EDTA reaches the bottom of the wells.
3. Add 60 L 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.
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.
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:
* 1400­2000 × g for 45 min
or
* 2000­3000 × g for 30 min
DRAFT
August 27, 2002 12:28 pm, 04_Purifying.fm
Chapter 4 Purifying Extension Products
4-4 BigDye
Terminator v3.1 Cycle Sequencing Kit
7. Invert the plate and spin up to 185 × g, then remove from the
centrifuge.
8. Add 60 L 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.
IMPORTANT! 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.
To precipitate 20-L sequencing reactions in 96-well reaction
plates: (continued)
DRAFT
August 27, 2002 12:28 pm, 04_Purifying.fm
Ethanol/EDTA Precipitation
BigDye
Terminator v3.1 Cycle Sequencing Kit 4-5
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-L sequencing reactions 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 L of 125 mM EDTA to each well.
Note: Make sure the EDTA reaches the bottom of the wells.
3. Add 25 L 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.
DRAFT
August 27, 2002 12:28 pm, 04_Purifying.fm
Chapter 4 Purifying Extension Products
4-6 BigDye
Terminator v3.1 Cycle Sequencing Kit
6.
IMPORTANT! Proceed to the next step immediately. If this is
not possible, then spin the plate for 2 minutes more
immediately before performing the next step.
7. Invert the plate and spin up to 185 × g, then remove from the
centrifuge.
8. Add 30 L 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.
IMPORTANT! 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.
To precipitate 10-L sequencing reactions in 384-well reaction
plates: (continued)
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:
* 1400­2000 × g for 45 min
or
* 2000­3000 × g for 30 min
DRAFT
August 27, 2002 12:28 pm, 04_Purifying.fm
Ethanol/EDTA/Sodium Acetate Precipitation
BigDye
Terminator v3.1 Cycle Sequencing Kit 4-7
Ethanol/EDTA/Sodium Acetate Precipitation
Note: Ethanol/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-L sequencing reactions in 96-well reaction
plates:
1. Remove the 96-well reaction plate from the thermal cycler
and briefly spin.
2. Add 2 L of 125 mM EDTA to each well.
Note: Make sure the EDTA reaches the bottom of the wells.
3. Add 2 L of 3 M sodium acetate to each well.
Note: Make sure the sodium acetate reaches the bottom of
the wells.
4. Add 50 L 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.
DRAFT
August 27, 2002 12:28 pm, 04_Purifying.fm
Chapter 4 Purifying Extension Products
4-8 BigDye
Terminator v3.1 Cycle Sequencing Kit
7.
IMPORTANT! Proceed to the next step immediately. If this is
not possible, then spin the plate for 2 minutes more
immediately before performing the next step.
8. Invert the plate and spin up to 185 × g, then remove from the
centrifuge.
9. Add 70 L 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.
IMPORTANT! 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.
To precipitate 20-L sequencing reactions in 96-well reaction
plates: (continued)
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:
* 1400­2000 × g for 45 min
or
* 2000­3000 × g for 30 min
DRAFT
August 27, 2002 12:28 pm, 04_Purifying.fm
Ethanol/EDTA/Sodium Acetate Precipitation
BigDye
Terminator v3.1 Cycle Sequencing Kit 4-9
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-L sequencing reactions in 384-well reaction
plates:
1. Remove the 384-well reaction plate from the thermal cycler
and briefly spin.
2. Add 1 L of 125 mM EDTA to each well.
Note: Make sure the EDTA reaches the bottom of the wells.
3. Add 1 L of 3 M sodium acetate to each well.
Note: Make sure the sodium acetate reaches the bottom of
the wells.
4. Add 25 L 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.
DRAFT
August 27, 2002 12:28 pm, 04_Purifying.fm
Chapter 4 Purifying Extension Products
4-10 BigDye
Terminator v3.1 Cycle Sequencing Kit
7.
IMPORTANT! Proceed to the next step immediately. If this is
not possible, then spin the plate for 2 minutes more
immediately before performing the next step.
8. Invert the plate and spin up to 185 × g, then remove from the
centrifuge.
9. Add 35 L 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.
IMPORTANT! 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.
To precipitate 10-L sequencing reactions in 384-well reaction
plates: (continued)
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:
* 1400­2000 × g for 45 min
or
* 2000­3000 × g for 30 min
DRAFT
August 27, 2002 12:28 pm, 04_Purifying.fm
Plate and Spin Column Purification
BigDye
Terminator v3.1 Cycle Sequencing Kit 4-11
Plate and Spin Column Purification
Overview This section provides instructions for adding a sodium dodecyl
sulfate (SDS)/heat treatment to the spin column and spin plate
purification methods. This SDS/heat treatment effectively eliminates
unincorporated dye terminators from cycle sequencing reactions.
Preparing Extension Products
Use this procedure to prepare extension products for both spin
column and 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 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 L of 2.2% SDS to each 20-L
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
DRAFT
August 27, 2002 12:28 pm, 04_Purifying.fm
Chapter 4 Purifying Extension Products
4-12 BigDye
Terminator v3.1 Cycle Sequencing Kit
Performing Spin Column Purification
Recommended
Spin Columns
We recommend Centri-SepTM spin columns (Applied Biosystems,
PN 401763 for 32 columns and PN 401762 for 100 columns).
Optimizing Spin
Column
Purification
IMPORTANT! When using the BigDye terminators v3.1, hydrate the
column for 2 hours (see page 4-13).
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 325­730 × 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.
5. Spin down the contents of the tubes briefly.
6. Continue with spin column or 96-well plate purification.
To prepare extension products: (continued)
DRAFT
August 27, 2002 12:28 pm, 04_Purifying.fm
Plate and Spin Column Purification
BigDye
Terminator v3.1 Cycle Sequencing Kit 4-13
Preparing the
Spin Column
To perform spin column purification:
1. Prepare the extension products according to "Preparing
Extension Products" on page 4-11.
2. Gently tap the column to cause the gel material to settle to
the bottom of the column.
3. Remove the upper end cap and add 0.8 mL of deionized
water.
4. Replace the upper end cap and vortex or invert the column a
few times to mix the water and gel material.
5. Allow the gel to hydrate at room temperature for at least 2
hours.
Note: Hydrated columns can be stored for a few days at
2­6 °C. Longer storage in water is not recommended. Allow
columns stored at 2­6 °C to warm to room temperature
before use.
6. Remove any air bubbles by inverting or tapping the column
and allowing the gel to settle.
7. 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.
8. Insert the column into the wash tube provided.
9. Spin the column in a microcentrifuge at 730 × g for
2 minutes to remove the interstitial fluid.
10. Remove the column from the wash tube and insert it into a
sample collection tube (for example, a 1.5-mL
microcentrifuge tube).
DRAFT
August 27, 2002 12:28 pm, 04_Purifying.fm
Chapter 4 Purifying Extension Products
4-14 BigDye
Terminator v3.1 Cycle Sequencing Kit
Purifying with the
Spin Column
Performing 96-Well Spin Plate Purification
Recommended
96-Well Spin
Plates
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.
Purifying with the
96-Well Spin
Plate
To perform purification with the spin column
1. Remove the extension reaction/SDS mixture from its tube
and load it carefully onto the center of the gel material.
2. 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.
3. Discard the column. The sample is in the sample collection
tube.
4. Dry the sample in a vacuum centrifuge for 10­15 minutes
without heat, or until dry. Do not over-dry.
To perform purification with the spin plate:
1. Prepare the extension products according to "Preparing
Extension Products" on page 4-11.
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.
DRAFT
August 19, 2002 3:58 pm, AppA_SelectingPrimers.fm
BigDye
Terminator v3.1 Cycle Sequencing Kit A-1
ASelecting Sequencing PrimersA
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 30­80%.
* 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.
DRAFT
August 19, 2002 3:58 pm, AppA_SelectingPrimers.fm
Appendix A Selecting Sequencing Primers
A-2 BigDye
Terminator v3.1 Cycle Sequencing Kit
DRAFT
August 19, 2002 3:56 pm, AppB_ControlSequence.fm
BigDye
Terminator v3.1 Cycle Sequencing Kit B-1
BControl DNA Sequence 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
DRAFT
August 19, 2002 3:56 pm, AppB_ControlSequence.fm
Appendix B Control DNA Sequence
B-2 BigDye
Terminator v3.1 Cycle Sequencing Kit
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
DRAFT
August 26, 2002 11:40 am, AppC_Electrophoresis.fm
BigDye
Terminator v3.1 Cycle Sequencing Kit C-1
CSample Electrophoresis C
Some Important Reminders
* Dye set/primer (mobility) files for the BigDye
terminators v3.1
are the same for those for v3.0, but different than those for the
dRhodamine terminators, BigDye terminators original, and
BigDye terminators v2.0.
* 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 "Dye Set/Primer (Mobility) Files" on page 1-6 for
information on obtaining the v3.1 dye set/primer (mobility) files.
DRAFT
August 26, 2002 11:40 am, AppC_Electrophoresis.fm
Appendix C Sample Electrophoresis
C-2 BigDye
Terminator v3.1 Cycle Sequencing Kit
Electrophoresis on the ABI PRISM 3700 DNA
Analyzer
Requirements Run Modules
Dye Set/Primer (Mobility) Files
Standards
IMPORTANT! Use Dye Set H.
Note: Refer to the product insert for instructions on using the
standards for this instrument.
Configuration Run Module
POP-5TM polymer, 50-cm Seq1_1POP5DefaultModule
Seq1_2POP5DefaultModule
POP-6TM polymer, 50-cm Seq1_1POP6DefaultModule
Seq1_2POP6DefaultModule
Polymer Dye Set/Primer (Mobility) File
POP-5 polymer DT3700POP5{BDv3}v1.mob
POP-6 polymer DT3700POP6{BDv3}v1.mob
Dye Set Standards
H 3700/3730 BigDye
Terminator v3.1 Sequencing
Standard (PN 4336943)
H 3700 BigDye
Terminator v3.1 Matrix Standard
(PN 4336975)
DRAFT
August 26, 2002 11:40 am, AppC_Electrophoresis.fm
Electrophoresis on the ABI PRISM 3700 DNA Analyzer
BigDye
Terminator v3.1 Cycle Sequencing Kit C-3
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)
DRAFT
August 26, 2002 11:40 am, AppC_Electrophoresis.fm
Appendix C Sample Electrophoresis
C-4 BigDye
Terminator v3.1 Cycle Sequencing Kit
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
Dye Set/Primer (Mobility) Files
Standards
IMPORTANT! Use Dye Set Z.
Note: Refer to the product insert for instructions on using the
standards for this instrument.
Configuration Run Module
POP-4TM
polymer, 36-cm UltraSeq_POP4Default Module
POP-4 polymer, 80-cm LongSeq80_POP4DefaultModule
POP-6TM
polymer, 36-cm RapidSeq36_POP6DefaultModule
POP-6 polymer, 50-cm StdSeq50_POP6DefaultModule
Polymer Dye Set/Primer (Mobility) File
POP-4TM
polymer DT3100POP4{BDv3}v1.mob
POP-6TM polymer DT3100POP6{BDv3}v1.mob
Dye Set Standards
Z BigDye
Terminator v3.1 Sequencing Standard
(PN 4336935)
Z 3100 BigDye
Terminator v3.1 Matrix Standard
(PN 4336974)
DRAFT
August 26, 2002 11:40 am, AppC_Electrophoresis.fm
Electrophoresis on the ABI PRISM 3100 and 3100-Avant Genetic Analyzers
BigDye
Terminator v3.1 Cycle Sequencing Kit C-5
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)
DRAFT
August 26, 2002 11:40 am, AppC_Electrophoresis.fm
Appendix C Sample Electrophoresis
C-6 BigDye
Terminator v3.1 Cycle Sequencing Kit
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
Dye Set/Primer (Mobility) Files
Configuration Run Module
POP-4TM polymer, 1-mL syringe,
47-cm, 50-m i.d. capillary, Ld = 36
cm
P4StdSeq (1 mL) E
POP-4 polymer, Rapid Sequencing,
1-mL syringe, 47-cm, 50-m i.d.
capillary, Ld = 36 cm
P4RapidSeq (1 mL) E
POP-6TM polymer, 1-mL syringe, 61cm,
50-m i.d. capillary
Seq POP6 (1 mL) E
POP-6 polymer, Rapid Sequencing,
1-mL syringe, 47-cm, 50-m i.d.
capillary
Seq POP6 Rapid (1 mL) E
Polymer Dye Set/Primer (Mobility) File
POP-4 polymer DT310POP4{BDv3}v2.mob
POP-6 polymer DT310POP6{BDv3}v2.mob
DRAFT
August 26, 2002 11:40 am, AppC_Electrophoresis.fm
Electrophoresis on the ABI PRISM 310 Genetic Analyzer
BigDye
Terminator v3.1 Cycle Sequencing Kit C-7
Matrix Standards
IMPORTANT! The instrument (matrix) file for the BigDye
terminators v3.1 cannot be used for the BigDye terminators
(original), BigDye terminators v2.0, dRhodamine terminators, or
BigDye
primers (original).
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)
Dye/Filter
Set
Standards for Instrument (Matrix) File Generation
E 310/377 BigDye
Terminator v3.1 Matrix Standards
(PN 4336948)
DRAFT
August 26, 2002 11:40 am, AppC_Electrophoresis.fm
Appendix C Sample Electrophoresis
C-8 BigDye
Terminator v3.1 Cycle Sequencing Kit
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
Dye Set/Primer (Mobility) Files
*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.
Configuration*
*Any plate check and prerun module can be used on the ABI PRISM 377 DNA
Sequencers.
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
Gel Formulation Dye Set/Primer (Mobility) File
4.5% acrylamide (29:1) or 5%
Long RangerTM gel
DT377{BDv3}v2.mob
DRAFT
August 26, 2002 11:40 am, AppC_Electrophoresis.fm
Electrophoresis on the ABI PRISM 377 DNA Sequencers
BigDye
Terminator v3.1 Cycle Sequencing Kit C-9
Matrix Standards
IMPORTANT! The instrument (matrix) file for the BigDye
terminators v3.1 cannot be used for the BigDye terminators
(original), BigDye terminators v2.0, dRhodamine terminators, or
BigDye primers (original), but can be used with BigDye terminators
v3.0.
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 v3.1 on the 377 instrument
in conjunction with the ABI PRISM
Lane GuideTM 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)
Dye/Filter
Set
Standards for Instrument (Matrix) File Generation
E 310/377 BigDye
Terminator v3.1 Matrix Standards
(PN 4336948)
DRAFT
August 26, 2002 11:40 am, AppC_Electrophoresis.fm
Appendix C Sample Electrophoresis
C-10 BigDye
Terminator v3.1 Cycle Sequencing Kit
DRAFT
August 27, 2002 10:01 am, AppD_Troubleshooting.fm
BigDye
Terminator v3.1 Cycle Sequencing Kit D-1
DTroubleshooting D
Observation Possible Causes Recommended Actions
No recognizable
sequence
Insufficient template ˇ Quantitate DNA template
* Increase amount of DNA in the sequencing
reactions
Inhibitory contaminant in
the template
Clean up the template
Insufficient primer ˇ 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
* Calibrate the thermal cycler regularly
* Use correct thermal cycling parameters
* Use correct tube for your thermal cycler
* Set ramp rate to 1 °C/sec
Extension products lost
during reaction cleanup
* Make sure you use the correct
centrifugation speeds and times for
precipitation and spin column procedures
* Make sure ethanol concentration is correct
for precipitation protocols
DRAFT
August 27, 2002 10:01 am, AppD_Troubleshooting.fm
Appendix D Troubleshooting
D-2 BigDye
Terminator v3.1 Cycle Sequencing Kit
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
Noisy data throughout
the sequence with low
signal strength
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
* Calibrate the thermal cycler regularly
* Use correct thermal cycling parameters
* Use correct tube for your thermal cycler
* Set ramp rate to 1 °C/sec
Lane tracking failure
(377 instrument)
Check the lane tracking. If necessary, retrack
and reextract the lanes
Electrokinetic injection
failure (capillary
instruments)
Repeat the injections
Noisy data throughout
the sequence with good
signal strength
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 ˇ 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
Observation Possible Causes Recommended Actions
DRAFT
August 27, 2002 10:01 am, AppD_Troubleshooting.fm
BigDye
Terminator v3.1 Cycle Sequencing Kit D-3
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
Noise up to or after a
specific point in the
sequence
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
Poor mobility correction 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
Observation Possible Causes Recommended Actions
DRAFT
August 27, 2002 10:01 am, AppD_Troubleshooting.fm
Appendix D Troubleshooting
D-4 BigDye
Terminator v3.1 Cycle Sequencing Kit
Excess dye peaks at
beginning of sequence
Poor removal of
unincorporated dye
terminators
Use ethanol/EDTA precipitation protocol to
remove unincorporated dye terminators.
When preparing extension products for plate
and spin column purification, increase the
final SDS concentration to 0.4%.
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
Observation Possible Causes Recommended Actions
DRAFT
August 19, 2002 3:57 pm, AppE_TechSupp.fm
BigDye
Terminator v3.1 Cycle Sequencing Kit E-1
EObtaining Technical Support 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.
DRAFT
August 19, 2002 3:57 pm, AppE_TechSupp.fm
Appendix E Obtaining Technical Support
E-2 BigDye
Terminator v3.1 Cycle Sequencing Kit
DRAFT
August 21, 2002 8:30 am, 4337035A_v3.1IX.fm
BigDye Terminator Cycle Sequencing Ready Reaction v3.1 Index-1
Index
A
attention words 1-11
B
bacterial artificial chromosomes 2-3
C
centrifuge, recommended 1-9
components, sequencing kits 1-6
contaminants, of templates 2-4
control
double-stranded 2-1
single-stranded 2-1
control DNA, reason for 2-1
customer support. See technical support E-1
D
disposables
cycle sequencing 1-8
purifying 1-9
DNA
determining quality 2-6
quantity to use 2-6
dye filter/set
377 1-4
dye set
3100 1-5
3700 1-5
dye set/primer files, downloading 1-6
dye/filter set
310 1-4
3100 1-5
3700 1-5
377 1-4
E
ethanol/EDTA precipitation
384-well 4-5
96-well 4-3
ethanol/EDTA/sodium acetate
384-well 4-9
96-well 4-7
F
formulation, new 1-1
freeze-thaw, limits 1-7
G
general requirements 1-2
H
hazards
chemical 1-11
waste 1-12
I
instruments
sequencing 1-3
thermal cyclers 1-3
DRAFT
August 21, 2002 8:30 am, 4337035A_v3.1IX.fm
Index-2 BigDye Terminator Cycle Sequencing Ready Reaction v3.1
M
matrix standards
310 1-4
377 1-4
general requirements 1-2
mobility files
downloading 1-6
existing 1-2
MSDSs, ordering 1-13
P
purifying
importance of 2-3
PCR by ultrafiltration 2-4
Q
quality problems, template 2-4
quality, examining DNA 2-5
quantitating DNA 2-6
R
ready reaction format 1-2
reagents
electrophoresis 1-10
purifying 1-9
required files
matrix 1-1, 1-2
spectral 1-1, 1-2
requirements
matrix standards 1-2
S
safety
chemical 1-11
guide 1-11
sequencing buffer
optimizing 1-2
sequencing kits, ordering 1-6
signal intensities, and DNA quantity 2-6
spectral standards
3100 1-5
3700 1-5
storage temperatures 1-7
T
technical support E-1
U
user attention words 1-11
W
warning symbols 1-11
Headquarters
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Phone: +1 650.638.5800
Toll Free (In North America): +1 800.345.5224
Fax: +1 650.638.5884
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scientists. Applera Corporation consists of the
Applied Biosystems and Celera Genomics
businesses.
Printed in USA, 09/2002
Part Number 4337035 Rev. A