BDT_.

book Page i Monday, December 13, 1999 1:54 PM

ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kits

Original and Version 2.0

Protocol

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Copyright 1999, PE Corporation Printed in the U.S.A. For Research Use Only. Not for use in diagnostic procedures. Notice to Purchaser: Limited License The purchase of the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit or the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit v2.0 include a limited, nontransferable, non-exclusive license (without the right to resell, repackage, or sublicense) under U.S. Patent 5,800,996 and corresponding foreign 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 this patent by PE Corporation. 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, PE Corporation, PE Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404. Patents are pending in countries outside the United States. Notice to Purchaser: Limited License The purchase price of this product includes a limited, nontransferable license under U.S. Patent 5,075,216 or its foreign counterparts, owned by Roche Molecular Systems, Inc. and F. Hoffmann-LaRoche Ltd. (Roche), to use only this amount of the product for DNA Sequencing and related processes described in said patent solely for the research and development activities of the purchaser. No license under these patents to use the PCR process is conveyed expressly or by implication to the purchaser by the purchase of this product. A license to use the PCR Process for certain research and development activities accompanies the purchase of certain reagents from licensed suppliers such as PE when used in conjunction with an Authorized Thermal Cycler, or is available from PE Corporation. Further information on purchasing licenses to practice PCR Process may be obtained by contacting Director of Licensing at PE Corporation, 850 Lincoln Centre Drive, Foster City, California 94404, or at Roche Molecular Systems, Inc., 1145 Atlantic Avenue, Alameda, California 94501. 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 a PE commercial automated sequencing machine or other authorized DNA sequencing machines that have been authorized for such use by PE Applied Biosystems of PE Corporation, or for manual DNA sequencing. No license is hereby granted for the use of this kit, or the reagents therein, in any other automated sequencing machine. Such sublicense 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 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, GeneScan, and MicroAmp are registered trademarks of PE Corporation. ABI, Applied Biosystems, BigDye, CATALYST, PE, POP, and POP-6 are trademarks of PE Corporation. AmpliTaq and GeneAmp are registered trademarks of Roche Molecular Systems, Inc. Centricon is a trademark of W. R. Grace and Co. Centri-Sep is a trademark of Princeton Separations, Inc. pGEM is a registered trademark of Promega Corporation. All other trademarks are the sole property of their respective owners. PE Corporation, formerly the Perkin-Elmer Corporation, is committed to providing the worlds leading technology and information for life scientists. PE Corporation consists of the PE Biosystems and Celera Genomics businesses. PE Biosystems comprises four divisionsApplied Biosystems, PE Informatics, PerSeptive Biosystems, and Tropix. PE SCIEX, which is managed through the PerSeptive Biosystems Division, is a joint venture between PE Corporation and SCIEX, the instrumentation technology division of MDS Inc.

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Contents
1 Introduction
Chapter Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1 In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1 Two Kits Available. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2 Protocol for Two Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2 Comparing the Two Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2 BigDye Terminator Ready Reaction Kits . . . . . . . . . . . . . . . . . . . . . . 1-2 Cycle Sequencing with AmpliTaq DNA Polymerase, FS. . . . . . . . . . 1-3 BigDye Terminators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3 Comparing Peak Height Patterns . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4 Dye Spectra. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-6 Instrument Platforms and Required Software . . . . . . . . . . . . . . . . . . . . . . . . 1-7 Instrument Platforms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-7 Thermal Cyclers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-7 Run Modules and Dye Set/Primer (Mobility) Files . . . . . . . . . . . . . . 1-8 Instrument (Matrix) File Required . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-9 Reagents and Storage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-10 Available Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-10 Description of Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-10 Storage and Use of the Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-10 Materials Supplied by the User . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-11 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-11 Materials for Cycle Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-11 Materials for Purifying Extension Products . . . . . . . . . . . . . . . . . . . 1-12 Materials for Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-13 Thermal Cycling Tubes Required . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-14 Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-15

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Documentation User Attention Words . . . . . . . . . . . . . . . . . . . . . . . 1-15 Ordering MSDSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-15 Chemical Hazard Warning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-16

2 Preparing the Templates

Chapter Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In This Chapter. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Control DNA Templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Using Control DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Control DNA Sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . An Additional Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Template Preparation Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Single- and Double-Stranded Templates . . . . . . . . . . . . . . . . . . . . . . BAC DNA Templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PCR Templates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Use of the Primer Island Transposition Kit. . . . . . . . . . . . . . . . . . . . . . . . . . Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . About Transposons. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inserting Articial Transposons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Template and Primer Quantities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Template Quantity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Template Volume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1 2-1 2-2 2-2 2-2 2-2 2-3 2-3 2-3 2-4 2-5 2-5 2-5 2-5 2-5 2-6 2-6 2-6 2-6

3 Performing Cycle Sequencing

Chapter Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In This Chapter. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sequencing Plasmids and PCR Products . . . . . . . . . . . . . . . . . . . . . . . . . . . Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sequencing Plasmids on the 3700 . . . . . . . . . . . . . . . . . . . . . . . . . . . Instruments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Preparing the Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-1 3-1 3-2 3-2 3-2 3-2 3-2

ii

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Cycle Sequencing on the GeneAmp 9700, 9600, or 2400 . . . . . . . . . 3-3 Cycle Sequencing on the TC1 or DNA Thermal Cycler 480 . . . . . . . 3-3 Sequencing BAC DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-5 Thermal Cyclers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-5 Sequencing BAC DNA on the 3700 . . . . . . . . . . . . . . . . . . . . . . . . . . 3-5 Preparing Sequencing Reactions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-5 Performing Cycle Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-6 Sequencing Bacterial Genomic DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-7 Thermal Cyclers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-7 Sequencing Bacterial Genomic DNA on the 3700 . . . . . . . . . . . . . . . 3-7 Preparing Sequencing Reactions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-7 Cycle Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-8 Sequencing on the CATALYST 800. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-9 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-9 Options for Sequencing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-9 Manual Ethanol Precipitation Required . . . . . . . . . . . . . . . . . . . . . . . 3-9 Sequencing on the ABI PRISM 877 ITC. . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-10 Predened Temperature Proles . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-10 Ethanol Precipitation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-10

4 Purifying Extension Products

Chapter Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-1 In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-1 Choosing a Method of Purication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-2 Purpose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-2 Spin Column vs. Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-2 Plate and Spin Column Purication. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-2 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-2 Recommended 384-Well Plate Columns . . . . . . . . . . . . . . . . . . . . . . 4-2 Recommended 96-Well Plate Columns . . . . . . . . . . . . . . . . . . . . . . . 4-2 Recommended Spin Columns. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-3 Optimizing Spin Column Purication . . . . . . . . . . . . . . . . . . . . . . . . 4-3 Performing Spin Column Purication . . . . . . . . . . . . . . . . . . . . . . . . 4-4 Isopropanol Precipitation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-5
iii

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Precipitating in 384-Well Plates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-5 Precipitating in 96-Well Plates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-5 Precipitating in Microcentrifuge Tubes . . . . . . . . . . . . . . . . . . . . . . . 4-7 Ethanol Precipitation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-9 Unincorporated Terminators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-9 Precipitating in 384-Well Plates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-9 Precipitating in 96-Well Plates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-9 Precipitating in Microcentrifuge Tubes . . . . . . . . . . . . . . . . . . . . . . 4-11 Ethanol/Sodium Acetate Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-13 Procedure Not for 3700 DNA Analyzer. . . . . . . . . . . . . . . . . . . . . . 4-13 Precipitating in 96-Well Plates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-13 Precipitating Microcentrifuge Tubes . . . . . . . . . . . . . . . . . . . . . . . . 4-15

5 Sample Electrophoresis
Chapter Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In This Chapter. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Electrophoresis on the ABI PRISM 3700 DNA Analyzer . . . . . . . . . . . . . . . Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Electrophoresis on the ABI PRISM 310 Genetic Analyzer . . . . . . . . . . . . . . Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Resuspending the Samples. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Electrophoresis on the ABI PRISM 377 Sequencers . . . . . . . . . . . . . . . . . . . Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Using the Lane Guide Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Using Long-Read Gel and Buffer Formulations . . . . . . . . . . . . . . . . Resuspending and Loading the Samples . . . . . . . . . . . . . . . . . . . . . . Electrophoresis on the ABI PRISM 373 with BigDye Filter Wheel . . . . . . . Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Resuspending and Loading the Samples . . . . . . . . . . . . . . . . . . . . . . 5-1 5-1 5-2 5-2 5-2 5-2 5-3 5-4 5-4 5-4 5-4 5-5 5-6 5-6 5-6

A Control DNA Sequence

Control Sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-1 Partial Sequence of pGEM-3Zf(+). . . . . . . . . . . . . . . . . . . . . . . . . . . A-1

iv

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B Technical Support
Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-1 To Reach Us on the Web . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-1 Hours for Telephone Technical Support . . . . . . . . . . . . . . . . . . . . . . .B-1 To Reach Us by Telephone or Fax in North America . . . . . . . . . . . . .B-1 Documents on Demand. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-2 To Reach Us by E-Mail. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-3

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vi

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Introduction
Chapter Summary
Topic Two Kits Available Instrument Platforms and Required Software Reagents and Storage Materials Supplied by the User Safety

1
See Page 1-2 1-7 1-10 1-11 1-15

In This Chapter The following topics are covered in this chapter:

Introduction 1-1

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Two Kits Available

Protocol for Two This protocol describes how to use the following kits: Kits o ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction
Kit o ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit v2.0

IMPORTANT The protocol is identical for both of the kits.

Comparing the The ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Two Kits Kit v2.0 contains the same components as the ABI PRISM BigDye
Terminator Cycle Sequencing Ready Reaction Kit (original kit). However, the ratio of dideoxy to deoxy terminators has been changed in the v2.0 kit. The new formulation distributes more signal to the longer DNA fragments. Reactions generated with the v2.0 kit show higher signal in longer fragments relative to shorter fragments. Recommended Use of Version 2 Kit The v2.0 kit can be used in place of the original kit on all of the sequencing platforms (see Instrument Platforms on page 1-7), but is primarily recommended for use with the ABI PRISM 3700 DNA Analyzer and the ABI PRISM 377 DNA Sequencer with 48-cm well-to-read. The v2.0 kit is very effective when following the extended read protocol described in Achieving Longer High Accuracy Reads on the 377 Sequencer (P/N 4315153).

BigDye Both the ABI PRISM BigDye Terminator Cycle Sequencing Ready Terminator Ready Reaction Kit and the ABI PRISM BigDye Terminator Cycle Sequencing Reaction Kits Ready Reaction Kit v2.0 provide AmpliTaq DNA Polymerase, FS,
BigDye terminators, and all the required components for the sequencing reaction.

In the Ready Reaction format, the dye terminators, deoxynucleoside triphosphates, AmpliTaq DNA Polymerase, FS, rTth pyrophosphatase (a component in AmpliTaq DNA Polymerase, FS), magnesium chloride, and buffer are premixed into a single tube of Ready Reaction Mix and are ready to use. These reagents are suitable for performing uorescence-based cycle sequencing reactions on single-stranded or double-stranded DNA templates, on polymerase chain reaction (PCR) fragments, and on large templates, e.g., BAC clones.
1-2 Introduction

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The dNTP mix includes dITP in place of dGTP to minimize band compressions. The dNTP mix also uses dUTP in place of dTTP. dUTP improves the incorporation of the T terminator and results in a better T pattern.

Cycle Sequencing with AmpliTaq DNA Polymerase, FS

Both kit formulations contain the sequencing enzyme AmpliTaq DNA Polymerase, FS. This enzyme is a variant of Thermus aquaticus DNA polymerase that contains a point mutation in the active site. This results in less discrimination against dideoxynucleotides. This enzyme also has a second mutation in the amino terminal domain that virtually eliminates the 53 nuclease activity of AmpliTaq DNA Polymerase. The enzyme has been formulated with a thermally stable inorganic pyrophosphatase to eliminate problems associated with pyrophosphorolysis. Cycle sequencing protocols that rely on the use of AmpliTaq DNA Polymerase, FS offer the following advantages over traditional sequencing methods: o o o o o Less hands-on operation No alkaline denaturation step required for double-stranded DNA Same protocol for both single- and double-stranded templates Less starting template needed More reproducible results

BigDye PE Biosystems has developed a set of dye terminators labeled with Terminators novel, high-sensitivity dyes. The dye structures contain a uorescein
donor dye, e.g., 6-carboxyuorescein (6-FAM), linked to a dichlororhodamine (dRhodamine) acceptor dye. The excitation maximum of each dye label is that of the uorescein donor, and the emission spectrum is that of the dRhodamine acceptor. See Dye Spectra on page 1-6. The donor dye is optimized to absorb the excitation energy of the argon ion laser in the PE Biosystems DNA sequencing instruments. The linker affords extremely efcient energy transfer (quantum efciency nearly 1.0, i.e., 100%) between the donor and acceptor dyes. The BigDye terminators are 23 times brighter than the rhodamine dye terminators when incorporated into cycle sequencing products.

Introduction 1-3

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The BigDye terminators are labeled with the following dRhodamine acceptor dyes:
Color of Raw Data on ABI PRISM 3700 or 310 Electropherograms Green Red Blue Black Color of Raw Data on ABI PRISM 377 or 373 Gel Image Green Red Blue Yellow

Terminator A C G T

Acceptor Dye dR6G dROX dR110 dTAMRA

Comparing Peak Data generated with dRhodamine dye terminators or BigDye Height Patterns terminators gives more even peak-height patterns than data generated
with rhodamine dye terminators. In particular, the weak G after A pattern characteristic of the rhodamine dye terminators is greatly reduced (Figure 1-1 through Figure 1-4 on page 1-5).

Figure 1-1 Region of pGEM-3Zf(+) sequenced with rhodamine dye terminators

1-4 Introduction

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Figure 1-2 Region of pGEM-3Zf(+) sequenced with dRhodamine terminators

Figure 1-3 Region of pGEM-3Zf(+) sequenced with BigDye terminators

Figure 1-4 Region of pGEM-3Zf(+) sequenced with BigDye terminators v2.0

Introduction 1-5

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Dye Spectra The normalized emission spectra of the dRhodamine dyes in the
BigDye terminators are shown below.

1-6 Introduction

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Instrument Platforms and Required Software

Instrument The ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Platforms Kits are for use with the following instruments:
o o o ABI PRISM 3700 DNA Analyzer ABI PRISM 310 Genetic Analyzer ABI PRISM 377 DNA Sequencers o ABI PRISM 377 ABI PRISM 377-18 ABI PRISM 377 with XL Upgrade ABI PRISM 377 with 96-Lane Upgrade ABI PRISM 373 ABI PRISM 373 with XL Upgrade

ABI PRISM 373 DNA Sequencers with BigDye Filter Wheel

These kits are designed for use with ABI PRISM 373 DNA Sequencers and ABI PRISM 373 DNA Sequencers with XL Upgrade on which the ABI PRISM BigDye Filter Wheel has been installed. Refer to the ABI PRISM BigDye Filter Wheel User Bulletin (P/N 4304367) for more information.

Thermal Cyclers This protocol has been optimized for all PE Biosystems thermal cyclers,
including: o o o o o GeneAmp PCR Systems 9700, 9600, and 2400 ABI PRISM 877 Integrated Thermal Cycler CATALYST 800 Molecular Biology LabStation DNA Thermal Cycler 480 DNA Thermal Cycler (TC1)

If you use a thermal cycler not manufactured by PE 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.

Introduction 1-7

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Run Modules and You must use Filter Set E run modules and dye set/primer (mobility) Dye Set/Primer les on all instrument platforms except the ABI PRISM 373 DNA (Mobility) Files Sequencer. Use Filter Set A on ABI PRISM 373 DNA Sequencers with
the ABI PRISM BigDye Filter Wheel.
IMPORTANT Users of the ABI PRISM 3700 DNA Analyzer refer to the ABI PRISM 3700 DNA Analyzer Users Manual (P/N 4306152) for information on run modules and dye set/primer (mobility) les.

o o

Run modules and dye set/primer (mobility) les are included in the current versions of data collection software. The run modules and dye set/primer (mobility) les can be downloaded from the Internet: http://www.pebio.com/ab/techsupp/softlib.html If you do not have access to the Internet, you can get the les from PE Biosystems Technical Support, or from your local eld applications specialist (call your local sales ofce for more information).

Run Modules o Run modules are the same as for the dRhodamine terminators and BigDye primers. Dye Set/Primer (Mobility) Files o You must install new dye set/primer (mobility) les for the BigDye terminators (original and version 2 kits). o Dye set/primer le names for the dRhodamine terminators are similar to those for the BigDye terminators. Their respective mobility les can be mistaken for each other easily. If a mobility le for the wrong sequencing chemistry is used, some bases will be miscalled. This is because in the dRhodamine chemistry C is labeled with dTAMRA and T is labeled with dROX, whereas in the BigDye terminator chemistry C is labeled with dROX BigDye and T is labeled with dTAMRA BigDye. In addition there are differences in the mobility shifts of dRhodamine terminators and BigDye Terminators.

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Instrument Data analysis requires a Filter Set E instrument (matrix) le: (Matrix) File IMPORTANT Users of the ABI PRISM 3700 DNA Analyzer refer to the Required ABI PRISM 3700 DNA Analyzer Users Manual (P/N 4306152) for information on
instrument (matrix) les.

For the ABI PRISM 310, 377, and 373 with BigDye Filter Wheel: o Instrument (matrix) les are the same for dRhodamine terminator chemistry and Big Dye terminator chemistries (original and version 2). Instrument (matrix) les are made using the ABI PRISM dRhodamine matrix standards (P/N 403047). Refer to the Automated DNA Sequencing Chemistry Guide (P/N 4305080; http://www.pebio.com/ab/techsupp/310.html) for information on creating instrument les.

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Reagents and Storage

Available Kits The following kits are available:
Kit ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit Number of Reactions 100 1000 5000 ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit v2.0 100 1000 5000 25,000 Part Number 4303149 4303150 4303151 4314414 4314415 4314416 4314849

Description of A description of the kit components is listed below. Reagents o Terminator Ready Reaction Mix:
o o A-Dye Terminator labeled with dichloro[R6G] C-Dye Terminator labeled with dichloro[ROX] G-Dye Terminator labeled with dichloro[R110] T-Dye Terminator labeled with dichloro[TAMRA] Deoxynucleoside triphosphates (dATP, dCTP, dITP, dUTP) AmpliTaq DNA Polymerase, FS MgCl2 Tris-HCl buffer, pH 9.0

pGEM-3Zf(+) double-stranded DNA Control Template, 0.2 g/L 21 M13 Control Primer (forward), 0.8 pmol/L

Storage and Use of Store the kits at 15 to 25 C. Before each use of either kit, allow the the Kits frozen stocks to thaw at room temperature (do not heat). Whenever
possible, thawed materials should be kept on ice during use.
IMPORTANT Mix each stock thoroughly and then centrifuge briey to collect all the liquid at the bottom of each tube.

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Materials Supplied by the User

Overview IMPORTANT This section describes materials that are required for sample
preparation. Refer to the instruments user manual for materials that are required for the operation of the instrument. Topic Materials for Cycle Sequencing Materials for Purifying Extension Products Materials for Electrophoresis See Page 1-11 1-12 1-13

Materials for Cycle Sequencing

ABI PRISM 3700 DNA Analyzer Material GeneAmp PCR Systems 9700 or 9600 Thermal cycling tubes, see page 1-14 Thermal Cycler, see page 1-7. Thermal cycling tubes, see page 1-14 Thermal Cycler, see 1-7. Thermal cycling tubes, see page 1-14 Supplier PE Biosystems PE Biosystems

ABI PRISM 310 Genetic Analyzer PE Biosystems PE Biosystems

ABI PRISM 377 or 373 with BigDye Filter Wheel PE Biosystems PE Biosystems

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Materials for Purifying Extension Products

ABI PRISM 3700 DNA Analyzer Material Choose one of the following: o 384-Well Plate Columns for Purication o 96-Well Plate Columns for Purication o Ethanol (EtOH), non-denatured, 95% o Isopropanol, 100% anhydrous Aluminum foil tape, adhesive-backed Choose one of the following: o Spin column, Centri-Sep, 1-mL 32 columns, 100 columns o Ethanol (EtOH), non-denatured, 95% o Isopropanol, 100% anhydrous o 75% Isopropanol o Ethanol, non-denatured, and 95% Sodium acetate (NaOAc), 3 M, pH 4.6 Aluminum foil tape, adhesive-backed Choose one of the following: o 96-Well Plate Columns for Purication o Spin column, Centri-Sep, 1-mL 32 columns, 100 columns o Ethanol (EtOH), non-denatured, 95% o Isopropanol, 100% anhydrous o 75% Isopropanol o Ethanol, non-denatured, and 95% Sodium acetate (NaOAc), 3 M, pH 4.6 Aluminum foil tape, adhesive-backed See page 4-2. PE Biosystems P/N 401763, P/N 401762 MLS MLS MLS MLS, and PE Biosystems (P/N 400320) 3M (Scotch Tape P/N 439)a PE Biosystems P/N 401763, P/N 401762 MLS MLS MLS MLS, and PE Biosystems (P/N 400320) 3M (Scotch Tape P/N 439)a See page 4-2. See page 4-2. MLS MLS 3M (Scotch Tape P/N 439)a Supplier

ABI PRISM 310 Genetic Analyzer

ABI PRISM 377 or 373 with BigDye Filter Wheel

a. Contact 3M in the USA at (800) 364-3577 for your local 3M representative. Use of other tapes may result in leakage or contamination of the sample.

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Materials for Electrophoresis

Material

ABI PRISM 3700 DNA Analyzer Supplier MLS MLS PE Biosystems (P/N 4311320) PE Biosystems (P/N 4305609) Choose one of the following: o Deionized water o 2-Pyrrolidinone o Hi-Di Formamide, 25-mL bottle Matrix Standard Set DS-01, dROX, dTAMRA, dR6G, dR110 Formamide EDTA ABI PRISM dRhodamine Matrix Standards Kit Formamide EDTA 25 mM EDTA with 50 mg/mL blue dextran ABI PRISM dRhodamine Matrix Standards Kit

ABI PRISM 310 Genetic Analyzer MLS MLS PE Biosystems (P/N 403047)

ABI PRISM 377 or 373 with BigDye Filter Wheel MLS MLS PE Biosystems (P/N 402055) PE Biosystems (P/N 403047)

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Thermal Cycling The table below shows several thermal cyclers, along with the Tubes Required appropriate plates and tubes for each.
Thermal Cycler GeneAmp PCR System 9700 Plate or Tube MicroAmp 384-Well Reaction Plate MicroAmp 96-Well Reaction Plate MicroAmp Reaction Tubes, 0.2-L GeneAmp PCR System 9600 MicroAmp 96-Well Reaction Plate MicroAmp Reaction Tubes, 0.2-L MicroAmp Caps, 12 or 8/strip GeneAmp PCR System 2400 DNA Thermal Cycler 480a MicroAmp Reaction Tubes, 0.2-L MicroAmp Caps, 12 or 8/strip GeneAmp Thin-Walled Reaction Tubes, 0.5-mL GeneAmp Thin-Walled Reaction Tubes with Flat Cap DNA Thermal Cycler (TC1) a GeneAmp Thin-Walled Reaction Tubes, 0.5-mL PE Biosystems Part Number 4305505 N801-0560 N801-0533 N801-0560 N801-0533 N801-0534 N801-0535 N801-0533 N801-0534 N801-0535 N801-0537 N801-0737 N801-0537

a. These thermal cyclers require mineral oil that can be obtained from PE Biosystems (P/N 0186-2302)

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Safety
Documentation Five user attention words appear in the text of all PE Biosystems user User Attention documentation. Each word implies a particular level of observation or Words action as follows.
Note This word is used to call attention to information. IMPORTANT This word calls attention to information that is necessary for correct use of the kit or instrument. CAUTION This word informs the user that damage to the instrument could occur if the user does not comply with the information. It also indicates a potentially hazardous situation that could result in minor or moderate injury to the user.

! WARNING ! This word informs the user that serious physical injury
or illness to the user or other persons could occur if these required precautions are not taken.

! DANGER ! Indicates an imminently hazardous situation that, if not

avoided, will result in death or serious injury.

Ordering MSDSs You can order free additional copies of MSDSs for chemicals
manufactured or distributed by PE Biosystems using the contact information below.
To order MSDSs... Over the Internet Then... Use www.pebio.com/techsupport a. Select MSDS Search button b. Enter keywords (or partial words), or a part number, or the MSDSs Documents on Demand index number c. Select Search d. Select the Adobe Acrobat symbol to view, print, or download the document, or check the box of the desired document and delivery method (fax or e-mail) By automated telephone service from any country By telephone in the United States Use Documents on Demand on page B-2.

Dial 1-800-327-3002, then press 1

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To order MSDSs... By telephone from Canada

Then... If you want ordering instructions in... English French

Then dial 1-800-668-6913 and... Press 1, then 2, then 1 again Press 2, then 2, then 1

By telephone from any other country

See the back cover of this protocol booklet.

For chemicals not manufactured or distributed by PE Biosystems, call the chemical manufacturer.

Chemical Hazard ! WARNING ! CHEMICAL HAZARD. Some of the chemicals used with Warning PE Biosystems instruments are potentially hazardous and can cause
injury, illness or death.

Read and understand the material safety data sheets (MSDSs) provided by the chemicals manufacturer before you store, handle, or work with any chemicals or hazardous materials. Minimize contact with and inhalation of chemicals. Wear appropriate personal protective equipment when handling chemicals (e.g., safety glasses, gloves, or clothing). Consult the listing in the MSDS. Do not leave chemical containers open. Use only with adequate ventilation. Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturers 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.

o o

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Preparing the Templates

Chapter Summary
Topic Control DNA Templates Template Preparation Methods Single- and Double-Stranded Templates BAC DNA Templates PCR Templates Use of the Primer Island Transposition Kit Template and Primer Quantities

2
See Page 2-2 2-3 2-3 2-3 2-4 2-5 2-6

In This Chapter The following topics are covered in this chapter:

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Control DNA Templates

Using Control Include a control DNA template as one of the templates in a set of DNA 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 We recommend M13mp18 as a single-stranded control and Sequence pGEM-3Zf(+) as a double-stranded control. All PE Biosystems DNA
sequencing kits provide pGEM control DNA. All dye terminator cycle sequencing kits include a 21 M13 control primer. 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 The BigDye Terminator Cycle Sequencing Standard (P/N 4304154) Control 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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Template Preparation Methods

Single- and Refer to the Automated DNA Sequencing Chemistry Guide Double-Stranded (P/N 4305080, http://www.pebio.com/ab/techsupp/310.html) for Templates information on preparing single- and double-stranded templates. BAC DNA With larger DNA targets such as bacterial articial chromosomes Templates (BACs), the quality of DNA template is important to the success of the
sequencing reaction. Two methods have given good sequencing results: o o Alkaline lysis1 Cesium chloride (CsCl) banding

Internet Addresses for BAC DNA Protocols For other BAC DNA preparation protocols, refer to the following Internet addresses: o o o Centre National de Squenage (CNS, or Gnoscope): http://www.cns.fr/externe/arabidopsis/protoBAC.html University of Oklahoma Advanced Center for Genome Technology: http://www.genome.ou.edu/DblAcetateProcV3.html Washington Univ School of Medicine Genome Sequencing Center: http://genome.wustl.edu/gsc/Protocols/BAC.shtml

Commercial Kits Commercial kits are also available for BAC DNA preparation: o o QIAGEN-tip 100 (QIAGEN: P/N 10043, 25 reactions; 10045, 100 reactions) QIAGEN-tip 500 (QIAGEN: P/N 10063, 25 reactions; 10065, 100 reactions)

1. Marra, M., Weinstock, L.A., and Mardis, E.R. 1996. End sequence determination from large insert cloning using energy transfer uorescent primers. Genomic Methods 6: 11181122.

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PCR Templates Cycle sequencing provides the most reproducible results for
sequencing PCR templates. Although PCR fragments can be difcult to denature with traditional sequencing methods, cycle sequencing provides several chances to denature and extend the template, which ensures 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 (P/N N930-2119). The protocol for using these columns is provided in Purifying PCR Fragments. Purifying PCR Fragments To purify PCR fragments by ultraltration:
Step 1 2 3 4 Action Assemble the Centricon-100 column according to the manufacturers recommendations. Load 2 mL deionized water onto the column. Add the entire sample to the column. Spin the column at 3000 g in a xed-angle centrifuge for 10 minutes. Note The manufacturer recommends a maximum speed of 1000 g, but 3000 g has worked well in PE Biosystems laboratories. If you are following the manufacturers guidelines, increase the time to compensate. 5 6 7 Remove the waste receptacle and attach the collection vial. Invert the column and spin it at 270 g for 2 minutes to collect the sample. This should yield approximately 4060 L of sample. Add deionized water to bring the puried PCR fragments to the original volume.

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Use of the Primer Island Transposition Kit

Overview BigDye terminators are also suitable for sequencing plasmid templates
generated using the Primer Island Transposition Kit (P/N 402984). This kit uses transposons to insert primer binding sites into cloned DNA.

About Transposons are mobile genetic elements, regions of DNA capable of Transposons inserting themselves (or copies of themselves) into the genome.
Transposons encode the proteins that facilitate their insertion into the target DNA.

Inserting Articial This property of transposons can be exploited to place unique primer Transposons binding sites randomly throughout any large segment of DNA. These
primer sites may be used subsequently as templates for PCR and/or sequencing reactions. Transposon insertion is an alternative to subcloning or primer walking when sequencing a large cloned DNA region.2,3 The Primer Island Transposition Kit provides reagents for generating articial transposon insertions into target DNA in vitro. The articial transposon contains the PI(+) and PI() priming sites. The Primer Island reagents are combined with a target DNA of choice and used to transform Escherichia coli.

Technique To identify the E. coli carrying the transposon, the transformed bacteria
are plated on Luria-Bertani (LB) agar plates containing carbenicillin and trimethoprim antibiotics. Each carbenicillin- and trimethoprim-resistant colony has integrated a copy of the transposon into the target DNA. Follow the Primer Island Transposition Kit Protocol (P/N 402920) for transposon insertion and template preparation.

2. Devine, S.E., and Boeke, J.D. 1994. Efcient integration of articial transposons into plasmid targets in vitro: a useful tool for DNA mapping, sequencing, and functional analysis. Nucleic Acids Res. 22: 37653772. 3. Devine, S.E., Chissoe, S.L., Eby, Y., Wilson, R.K., and Boeke, J.D. 1997. A transposon-based strategy for sequencing repetitive DNA in eukaryotic genomes. Genome Res. 7: 551563.

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Template and Primer Quantities

Overview If possible, quantitate the amount of puried 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.
Template PCR product: 100200 bp 200500 bp 5001000 bp 10002000 bp >2000 bp Single-stranded Double-stranded Cosmid, BAC Bacterial genomic DNA Note 13 ng 310 ng 520 ng 1040 ng 40100 ng 50100 ng 200500 ng 0.51.0 g 23 g Quantity

In general, higher DNA quantities give higher signal intensities.

Note The template quantities stated above should work with all primers. you may be able to use even less DNA, especially when sequencing with the 21 M13 primer. The amount of PCR product to use in sequencing will also depend on the length and purity of the PCR product.

Template Volume Cycle-sequencing reactions are made up in a nal volume of 20 L. The

volume includes 8 L for DNA template and 4 L for primer. If your DNA is not concentrated enough and you need to add more than 8 L of DNA template, then you can compensate for the additional volume by using a more concentrated solution of primer. For example, if your concentration of primers is increased from 0.8 pmol/L to 3.2 pmol/L, then the volume of primers can be reduced from 4 L to 1 L. Because less volume is used for the primers, more volume can then be added for the template. In this example, the volume of DNA template could be increased from 8 L to 11 L.

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Performing Cycle Sequencing

Chapter Summary
Topic Sequencing Plasmids and PCR Products Sequencing BAC DNA Sequencing Bacterial Genomic DNA Sequencing on the Catalyst 800 Sequencing on the ABI Prism 877 ITC

3
See Page 3-2 3-5 3-7 3-9 3-10

In This Chapter The following topics are covered in this chapter:

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Sequencing Plasmids and PCR Products

Overview This section describes how to prepare reactions and perform cycle
sequencing on plasmids and PCR Products.

Sequencing IMPORTANT If you are sequencing plasmids and PCR products on the Plasmids on the ABI PRISM 3700 DNA Analyzer, refer to the ABI PRISM 3700 DNA Analyzer 3700 Sequencing Chemistry Guide (P/N 4309125) for information about reaction set
up and cycle sequencing.

Instruments The following thermal cyclers can be used with this protocol:
o o o o o GeneAmp PCR Systems 9700, 9600, and 2400 ABI PRISM 877 Integrated Thermal Cycler CATALYST 800 Molecular Biology LabStation DNA Thermal Cycler 480 DNA Thermal Cycler (TC1)

Preparing the The type of tube required depends on the thermal cycler that you are Reactions using. Refer to Thermal Cycling Tubes Required on page 1-14.
To prepare the reaction mixtures:
Step 1 Action For each reaction add the following reagents to a separate tube: Reagent Terminator Ready Reaction Mix Template Single-stranded DNA Double-stranded DNA PCR product DNA Primer Deionized water Total Volume 2 Mix well and spin briey. 50100 ng 200500 ng See table in Template Quantity on page 2-6. 3.2 pmol Quantity 8.0 L

q.s.
20 L

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To prepare the reaction mixtures: (continued)

Step 3 Action If using the DNA Thermal Cycler (TC1) or DNA Thermal Cycler 480, overlay reaction mixture with 40 L of light mineral oil.

Cycle Sequencing To sequence DNA on the GeneAmp PCR System 9700, 9600, or 2400: on the GeneAmp Step Action 9700, 9600, or 2400
1 2 Place the tubes in a thermal cycler, and set the volume to 20 L. Repeat the following for 25 cycles: o Rapid thermal rampa to 96 C o 96 C for 10 seconds. o Rapid thermal ramp to 50 C o 50 C for 5 seconds. o Rapid thermal ramp to 60 C o 60 C for 4 minutes. 3 4 5 Rapid thermal ramp to 4 C and hold until ready to purify. Spin down the contents of the tubes in a microcentrifuge. Proceed to Chapter 4, Purifying Extension Products.

a. Rapid thermal ramp is 1 C/sec.

Cycle Sequencing To sequence DNA on the TC1 or DNA Thermal Cycler 480: on the TC1 or Step Action DNA Thermal 1 Place the tubes in a thermal cycler, and set the volume to 20 L. Cycler 480
2 Repeat the following for 25 cycles: o Rapid thermal rampa to 96 C o 96 C for 30 seconds. o Rapid thermal ramp to 50 C o 50 C for 15 seconds. o Rapid thermal ramp to 60 C o 60 C for 4 minutes. 3 4 Rapid thermal ramp to 4 C and hold until ready to purify. Spin down the contents of the tubes in a microcentrifuge.

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To sequence DNA on the TC1 or DNA Thermal Cycler 480:

Step 5 Action Proceed to Chapter 4, Purifying Extension Products.

a. Rapid thermal ramp is 1 C/sec.

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Sequencing BAC DNA

Thermal Cyclers The following thermal cyclers can be used with this protocol:
o o o GeneAmp PCR Systems 9600 or 9700 (in 9600 emulation mode) ABI PRISM 877 Integrated Thermal Cycler CATALYST 800 Molecular Biology LabStation

This protocol needs to be reoptimized for use on other thermal cyclers.

Sequencing BAC IMPORTANT If you are sequencing BAC DNA on the ABI PRISM 3700 DNA DNA on the 3700 Analyzer, refer to the ABI PRISM 3700 DNA Analyzer Sequencing Chemistry
Guide (P/N 4309125) for information about reaction set up and cycle sequencing.

Preparing The type of tube required depends on the thermal cycler that you are Sequencing using. Refer to Thermal Cycling Tubes Required on page 1-14. Reactions To prepare the sequencing reaction:
Step 1 Action For each reaction, add the following reagents to a separate tube: Reagent Terminator Ready Reaction Mix DNA Template Primer Deionized water Total Volume 2 Mix well and spin briey. Quantity 16 L 0.51.0 g 510 pmol

q.s.
40 L

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Performing Cycle To perform cycle sequencing on BAC DNA: Sequencing

Step 1 2 3 Action Place the tubes in a thermal cycler and set the volume to 30 L. Heat the tubes at 95 C for 5 minutes. Repeat the following for 30 cycles:a o Rapid thermal rampb to 95 C o 95 C for 30 seconds. o Rapid thermal ramp to 5055 C (depending on template) o 5055 C for 10 seconds. o Rapid thermal ramp to 60 C o 60 C for 4 minutes. 4 5 6 Rapid thermal ramp to 4 C and hold until ready to purify. Spin down the contents of the tubes in a microcentrifuge. Proceed to Chapter 4, Purifying Extension Products.

a. Some laboratories have found that increasing the number of cycles gives better results. b. Rapid thermal ramp is 1 C/sec.

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Sequencing Bacterial Genomic DNA

Thermal Cyclers The following thermal cyclers can be used with this protocol.This
protocol needs to be reoptimized for use on other thermal cyclers. o o o GeneAmp PCR Systems 9600 or 9700 (in 9600 emulation mode) ABI PRISM 877 Integrated Thermal Cycler CATALYST 800 Molecular Biology LabStation

Sequencing IMPORTANT If you are sequencing bacterial genomic DNA on the ABI PRISM Bacterial Genomic 3700 DNA Analyzer, refer to the ABI PRISM 3700 DNA Analyzer Sequencing DNA on the 3700 Chemistry Guide (P/N 4309125) for information about reaction set up and cycle
sequencing.

Preparing The type of tube required depends on the thermal cycler that you are Sequencing using. Refer to Thermal Cycling Tubes Required on page 1-14. Reactions To prepare the sequencing reactions for bacterial genomic DNA:
Step 1 Action For each reaction, add the following reagents to a separate tube: Reagent Terminator Ready Reaction Mix DNA Templatea Primer Deionized water Total Volume Quantity 16 L 23 g 613 pmol

q.s.
40 L

a. Shearing the DNA by passing it seven times through a 21-gauge, 1-inch long needle can improve signals.

Mix well and spin briey.

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Cycle Sequencing To perform cycle sequencing:

Step 1 2 3 Action Place the tubes in a thermal cycler, and set the volume to 40 L. Heat the tubes at 95 C for 5 minutes. Repeat the following for 45 cycles: o Rapid thermal rampa to 95 C o 95 C for 30 seconds. o Rapid thermal ramp to 5055 C (depending on template) o 55 C for 20 seconds. o Rapid thermal ramp to 60 C o 60 C for 4 minutes. 4 5 6 Rapid thermal ramp to 4 C and hold until ready to purify. Spin down the contents of the tubes in a microcentrifuge. Proceed to Chapter 4, Purifying Extension Products.

a. Rapid thermal ramp is 1 C/sec.

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Sequencing on the CATALYST 800

Overview Templates that have been prepared as described in chapter 2 should be
suitable for use on the CATALYST 800 Molecular Biology LabStation. Follow the protocols in the Turbo Appendix of the CATALYST 800 Molecular Biology LabStation Users Manual (P/N 903939) to set up your reactions.

Options for Terminator sequencing has two options: Sequencing o A reaction premix containing the sequencing primer or premixing
template with primer in the sample tube o A reaction cocktail (lacking primers), water, and primer from one tube combined with template from another tube

Manual Ethanol Ethanol precipitation is not available for Terminator Sequencing Precipitation protocols on the CATALYST 800 Molecular Biology LabStation. Ethanol Required precipitation or spin-column purication must be performed manually.
See Chapter 4, Purifying Extension Products.

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Sequencing on the ABI PRISM 877 ITC

Predened Predened temperature proles are provided for the following on the Temperature ABI PRISM 877 Integrated Thermal Cycler: Proles o Terminator Sequencing uses a reaction premix containing the
sequencing primer, or requires premixing template with primer in the sample tube. o Terminator Automix Sequencing combines reaction cocktail (lacking primers), water, primer from one tube, and template from another tube.

The prole is chosen on the Chemistry page of the Sequencing Notebook and can be edited to make custom proles. Refer to Chapter 4, Using the ABI PRISM 877 Software, in the ABI PRISM 877 Integrated Thermal Cycler Users Manual (P/N 904414).

Ethanol Ethanol precipitation can be chosen for dye terminator sequencing. The Precipitation proportions of ethanol and precipitation additive are set for default
reaction volumes. These volumes can be changed, especially if the reaction volume is modied. After the program is completed, proceed to Chapter 4, Purifying Extension Products. On extended runs (e.g., overnight), we recommend withholding addition of ethanol until plate processing can be completed. This delay can be programmed on the Chemistry page of the Sequencing Notebook.

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Purifying Extension Products 4

Chapter Summary
In This Chapter The following topics are covered in this chapter:
Topic Choosing a Method of Purication Plate and Spin Column Purication Isopropanol Precipitation Ethanol Precipitation Ethanol/Sodium Acetate Precipitation

4
See Page 4-2 4-2 4-5 4-9 4-13

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Choosing a Method of Purication

Purpose Unincorporated dye terminators must be completely removed before
the samples can be analyzed by electrophoresis. Excess dye terminators in sequencing reactions obscure data in the early part of the sequence and can interfere with base calling.

Spin Column vs. Use the method that works best for your particular application. Precipitation o Precipitation methods are cheaper and faster, but they remove less
of the unincorporated dye-labeled terminators that can obscure data at the beginning of the sequence. o The plate column and spin column procedures remove more terminators, but are more costly and take time to perform.

Plate and Spin Column Purication

Overview This section describes the recommended plate and spin columns for
purifying extension products.

Recommended For large-scale procedures, you can use the following commercially 384-Well Plate available 384-well reaction plate: Columns o ArrayIt (Telechem, P/N DTC-384-100)
o 384 System I (Edge Biosystems, P/N 95674)

Refer to the manufacturers instructions for procedures.

Recommended For large-scale procedures, you can use the following commercially 96-Well Plate available 96-well purication plates: Columns o 96-Well Spin Columns, Gel Filtration Kit (Edge Biosystems,
P/N 94880) o o o ArrayIt (Telechem, P/N DTC-96-100) Centri-Sep 96 plate (Princeton Separations, P/N CS-961) Multiscreen 96-Well Filter Plates (Millipore, P/N MADYEKIT1)

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Quantum Prep SEQueaky Kleen 96-well Terminator Removal Kit (Bio-Rad 732-6260)

Refer to the manufacturers instructions procedures.

Recommended We recommend Centri-Sep spin columns (Princeton Spin Columns Separations P/N CS-901). Optimizing Spin IMPORTANT For the BigDye terminators, hydrate the column for 2 hours. Column Tips for optimizing spin column purication: Purication
o o o Use one column for each sample. Do not process more columns than you can handle conveniently at one time. Load the sample in the center of the column bed. Make sure that the sample does not touch the sides of the column and that the pipet tip does not touch the gel surface. If samples are not properly loaded, peaks from unincorporated dye terminators can result. o 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 o o 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.

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Performing Spin To perform spin column purication: Column Step Action Purication
1 2 3 4 Gently tap the column to cause the gel material to settle to the bottom of the column. Remove the upper end cap and add 0.8 mL of deionized water. Replace the upper end cap and vortex or invert the column a few times to mix the water and gel material. 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. 5 6 Remove any air bubbles by inverting or tapping the column and allowing the gel to settle. Remove the upper end cap rst, then remove the bottom cap. Allow the column to drain completely by gravity. Note If ow does not begin immediately, apply gentle pressure to the column with a pipette bulb. 7 8 9 10 Insert the column into the wash tube provided. Spin the column in a microcentrifuge at 730 g for 2 minutes to remove the interstitial uid. Remove the column from the wash tube, and insert it into a sample collection tube (e.g., a 1.5-mL microcentrifuge tube). Remove the extension reaction mixture from its tube, and load it carefully onto the center of the gel material. Note If the TC1 or DNA Thermal Cycler 480 was used for thermal cycling, remove the reactions from the tubes as shown in step 1 on page 4-7. 11 Spin the column in a microcentrifuge at 730 g for 2 minutes. Note If using a centrifuge with a xed-angle rotor, place the column in the same orientation as it was in for the rst spin. This is important because the surface of the gel will be at an angle in the column after the rst spin. 12 13 Discard the column. The sample is in the sample collection tube. Dry the sample in a vacuum centrifuge for 1015 minutes, or until dry. Do not overdry.

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Isopropanol Precipitation
Precipitating in IMPORTANT If you are precipitating in 384-well plates, refer to the ABI Prism 384-Well Plates 3700 DNA Analyzer Sequencing Chemistry Guide (P/N 4309125) for the
procedure.

Precipitating in Note This procedure does not use salt. 96-Well Plates To precipitate in 96-Well MicroAmp Reaction Plates:
Step 1 2 Action Remove the MicroAmp Tray from the thermal cycler. Remove the caps from each tube. Add one of the following: o 80 L of 75% isopropanol o 20 L of deionized water and 60 L of 100% isopropanol The nal isopropanol concentration should be 60 5%.

! WARNING ! CHEMICAL HAZARD. Isopropyl alcohol can

be harmful if inhaled, ingested, or absorbed through the skin. It can cause CNS depression, and be irritating to the eyes, skin, and mucous membranes. Always work in a fume hood. Obtain a copy of the MSDS from the manufacturer. Wear appropriate protective eyewear, clothing, and gloves. 3 Seal the tubes with strip caps or by applying a piece of 3M Scotch Tape 439 adhesive-backed aluminum foil tape. Press the foil onto the tubes to prevent any leakage. Invert the tray a few times to mix. Leave the tray at room temperature for 15 minutes to precipitate the extension products. Note Precipitation times shorter than 15 minutes will result in the loss of very short extension products. Precipitation times longer than 24 hours will increase the precipitation of unincorporated dye terminators.

4 5

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To precipitate in 96-Well MicroAmp Reaction Plates: (continued)

Step 6 Action Place the tray in a table-top centrifuge with tube-tray adaptor and spin it at the maximum speed, which must be 1400 g but <3000 g: o 14002000 g: 45 minutes o 20003000 g: 30 minutes Note A MicroAmp tube in a MicroAmp Tray can withstand 3000 g for 30 minutes. IMPORTANT Proceed to the next step immediately. If not possible, then spin the tubes for 2 minutes more immediately before performing the next step. 7 Without disturbing the precipitates, remove the adhesive tape and discard the supernatant by inverting the tray onto a paper towel folded to the size of the tray. If you are performing this procedure for electrophoresis on the 3700 DNA Analyzer: a. Rinse the pellet by adding 150 L of 70% isopropanol to each well. b. Seal the plate with adhesive tape. c. Invert the plate a few times. 9 10 Place the inverted tray with the towel into the table-top centrifuge and spin at 700 g for 1 minute. Remove the tray and discard the paper towel. Note Pellets may or may not be visible. Vacuum drying of the samples is not necessary.

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Precipitating in To precipitate in microcentrifuge tubes: Microcentrifuge Step Action Tubes

1 Pipet the entire contents of each extension reaction into a 1.5-mL microcentrifuge tube. To remove reactions run on the TC1 or DNA Thermal Cycler 480: Place the pipette tip into the bottom of the reaction and carefully remove the reaction from the oil.

Oil Reaction IMPORTANT Transfer as little oil as possible. 2 Add one of the following: o 80 L of 75% isopropanol o 20 L of deionized water and 60 L of 100% isopropanol The nal isopropanol concentration should be 60 5%.

! WARNING ! CHEMICAL HAZARD. Isopropyl alcohol can

be harmful if inhaled, ingested, or absorbed through the skin. It can cause CNS depression, and be irritating to the eyes, skin, and mucous membranes. Always work in a fume hood. Obtain a copy of the MSDS from the manufacturer. Wear appropriate protective eyewear, clothing, and gloves. 3 4 Close the tubes and vortex briey. Leave the tubes at room temperature for 15 minutes to precipitate the extension products. Note Precipitation times shorter than 15 minutes will result in the loss of very short extension products. Precipitation times longer than 24 hours will increase the precipitation of unincorporated dye terminators. 5 Place the tubes in a microcentrifuge and mark their orientations. Spin the tubes for 20 minutes at maximum speed. IMPORTANT Proceed to the next step immediately. If not possible, then spin the tubes for 2 minutes more immediately before performing the next step.

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To precipitate in microcentrifuge tubes: (continued)

Step 6 Action Carefully aspirate the supernatants with a separate pipette tip for each sample and discard. Pellets may or may not be visible. IMPORTANT The supernatants must be removed completely, as unincorporated dye terminators are dissolved in them. The more residual supernatant left in the tubes, the more unincorporated dye terminators will remain in the samples. 7 8 9 10 Add 250 L of 75% isopropanol to the tubes, and vortex them briey. Place the tubes in the microcentrifuge in the same orientation as in step 5, and spin for 5 minutes at maximum speed. Aspirate the supernatants carefully, as in step 6. Dry the samples in a vacuum centrifuge for 1015 minutes or to dryness. (Alternatively, place the tubes with the lids open in a heat block or thermal cycler at 90 C for 1 minute.)

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Ethanol Precipitation
Unincorporated With ethanol precipitation, traces of unincorporated terminators may be Terminators seen at the beginning of the sequence data (up to base 40), but this is
usually minimal. Some loss in the recovery of the smallest fragments may also be observed.

Precipitating in IMPORTANT If you are precipitating in 384-well plates, refer to the ABI PRISM 384-Well Plates 3700 DNA Analyzer Sequencing Chemistry Guide (P/N 4309125) for the
procedure.

Precipitating in IMPORTANT Where 95% ethanol is recommended in precipitation protocols, 96-Well Plates purchase non-denatured ethanol at this concentration rather than absolute
(100%) ethanol. Absolute ethanol absorbs water from the atmosphere, gradually decreasing its concentration. This can lead to inaccurate nal concentrations of ethanol, which can affect some protocols.

To precipitate in 96-well MicroAmp plates:

Step 1 2 Action Remove the MicroAmp plate from the thermal cycler. Remove the caps from each tube. Add the following: o 16 L of deionized water o 64 L of non-denatured 95% ethanol The nal ethanol concentration should be 60 3%.

! WARNING ! CHEMICAL HAZARD. Ethanol is a

ammable chemical and is irritating to the skin, eyes, respiratory system. It can cause nerve and liver damage, CNS depression, nausea, vomiting, and headache. Always work in a fume hood. Obtain a copy of the MSDS from the manufacturer. Wear appropriate protective eyewear, clothing, and gloves. 3 Seal the tubes with strip caps or by applying a piece of 3M Scotch Tape 439 adhesive-backed aluminum foil tape. Press the foil onto the tubes to prevent any leakage. Invert the tray a few times to mix.

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To precipitate in 96-well MicroAmp plates: (continued)

Step 5 Action Leave the tray at room temperature for 15 minutes to precipitate the extension products. Note Precipitation times shorter than 15 minutes will result in the loss of very short extension products. Precipitation times longer than 24 hours will increase the precipitation of unincorporated dye terminators. 6 Place the tray in a tabletop centrifuge with tube-tray adaptor and spin it at the maximum speed, which must be 1400 g but <3000 g: o 14002000 g: 45 minutes o 20003000 g: 30 minutes Note A MicroAmp tube in a MicroAmp Tray can withstand 3000 g for 30 minutes. IMPORTANT Proceed to the next step immediately. If not possible, then spin the tubes for 2 minutes more immediately before performing the next step. 7 Without disturbing the precipitates, remove the adhesive tape and discard the supernatant by inverting the tray onto a paper towel folded to the size of the tray. If you are performing this procedure for electrophoresis on the 3700 DNA Analyzer: a. Rinse the pellet by adding 150 L of 70% ethanol to each well. b. Seal the plate with adhesive tape. c. Invert the plate a few times. 9 10 Place the inverted tray with the towel into the tabletop centrifuge, and spin at 700 g for 1 minute. Remove the tray and discard the paper towel. Note Pellets may or may not be visible. Vacuum drying of the samples is not necessary.

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Precipitating in To precipitate in microcentrifuge tubes: Microcentrifuge Step Action Tubes

1 Pipet the entire contents of each extension reaction into a 1.5-mL microcentrifuge tube. Note If the TC1 or DNA Thermal Cycler 480 was used for thermal cycling, remove the reactions from the tubes as shown in step 1 on page 4-7. 2 Add the following: o 16 L of deionized water o 64 L of non-denatured 95% ethanol The nal ethanol concentration should be 60 3%.

! WARNING ! CHEMICAL HAZARD. Ethanol is a

ammable chemical and is irritating to the skin, eyes, respiratory system. It can cause nerve and liver damage, CNS depression, nausea, vomiting, and headache. Always work in a fume hood. Obtain a copy of the MSDS from the manufacturer. Wear appropriate protective eyewear, clothing, and gloves. 3 4 Close the tubes and vortex briey. Leave the tubes at room temperature for 15 minutes to precipitate the extension products. Note Precipitation times shorter than 15 minutes will result in the loss of very short extension products. Precipitation times longer than 24 hours will increase the precipitation of unincorporated dye terminators. 5 Place the tubes in a microcentrifuge and mark their orientations. Spin the tubes for 20 minutes at maximum speed. IMPORTANT Proceed to the next step immediately. If not possible, then spin the tubes for 2 minutes more immediately before performing the next step. 6 Carefully aspirate the supernatants with a separate pipette tip for each sample and discard. Pellets may or may not be visible. IMPORTANT The supernatants must be removed completely, as unincorporated dye terminators are dissolved in them. The more residual supernatant left in the tubes, the more unincorporated dye terminators will remain in the samples. 7 Add 250 L of 70% ethanol to the tubes and vortex them briey.

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To precipitate in microcentrifuge tubes: (continued)

Step 8 9 10 Action Place the tubes in the microcentrifuge in the same orientation as in step 5 and spin for 10 minutes at maximum speed. Aspirate the supernatants carefully as in step 6. Dry the samples in a vacuum centrifuge for 1015 minutes or to dryness. (Alternatively, place the tubes with the lids open in a heat block or thermal cycler at 90 C for 1 minute.)

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Ethanol/Sodium Acetate Precipitation

Procedure Not for IMPORTANT This procedure is not recommended for use on the ABI PRISM 3700 DNA 3700 DNA Analyzer. Analyzer Precipitating in IMPORTANT Use non-denatured 95% ethanol rather than absolute (100%) 96-Well Plates ethanol. Absolute ethanol absorbs water from the atmosphere, gradually

decreasing its concentration. This can lead to inaccurate nal concentrations of ethanol, which can affect some protocols.

To precipitate in 96-well MicroAmp trays:

Step 1 2 Action Remove the MicroAmp Tray from the thermal cycler. Remove the caps from each tube. Add the following: o 2.0 L of 3 M sodium acetate (NaOAc), pH 4.6 o 50 L of 95% ethanol (EtOH) The nal ethanol concentration should be 65%.

! WARNING ! CHEMICAL HAZARD. Ethanol is a

ammable chemical and is irritating to the skin, eyes, respiratory system. It can cause nerve and liver damage, CNS depression, nausea, vomiting, and headache. Always work in a fume hood. Obtain a copy of the MSDS from the manufacturer. Wear appropriate protective eyewear, clothing, and gloves. 3 Seal the tubes with strip caps or by applying a piece of 3M Scotch Tape 425-3 adhesive-backed aluminum foil tape. Press the foil onto the tubes to prevent any leakage. Invert the tray a few times to mix. Leave the tray at room temperature for 15 minutes to precipitate the extension products. Note Precipitation times shorter than 15 minutes will result in the loss of very short extension products. Precipitation times longer than 24 hours will increase the precipitation of unincorporated dye terminators.

4 5

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To precipitate in 96-well MicroAmp trays: (continued)

Step 6 Action Place the tray in a tabletop centrifuge with tube-tray adaptor and spin it at the maximum speed, which must be 1400 g but <3000 g: o 14002000 g: 45 minutes o 20003000 g: 30 minutes Note A MicroAmp tube in a MicroAmp Tray can withstand 3000 g for 30 minutes. IMPORTANT Proceed to the next step immediately. If not possible, then spin the tubes for 2 minutes more immediately before performing the next step. 7 Without disturbing the precipitates, remove the adhesive tape and discard the supernatant by inverting the tray onto a paper towel folded to the size of the tray. Place the inverted tray with the towel into the table-top centrifuge and spin at 700 g for 1 minute. Add 150 L of 70% ethanol to each pellet. Cap or seal the tubes, then invert the tray a few times to mix. Spin the tray for 10 minutes at maximum speed. Repeat steps 7 and 8. Remove the tray and discard the paper towel. Note Pellets may or may not be visible. Vacuum drying of the samples is not necessary.

8 9 10 11 12 13

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Precipitating To precipitate in microcentrifuge tubes: Microcentrifuge Step Action Tubes

1 For each sequencing reaction, prepare a 1.5-mL microcentrifuge tube containing the following: o 2.0 L of 3 M sodium acetate (NaOAc), pH 4.6 o 50 L of 95% ethanol (EtOH) Note If the TC1 or DNA Thermal Cycler 480 was used for thermal cycling, remove the reactions from the tubes as shown in step 1 on page 4-7.

! WARNING ! CHEMICAL HAZARD. Ethanol is a

ammable chemical and is irritating to the skin, eyes, respiratory system. It can cause nerve and liver damage, CNS depression, nausea, vomiting, and headache. Always work in a fume hood. Obtain a copy of the MSDS from the manufacturer. Wear appropriate protective eyewear, clothing, and gloves. 2 3 Pipet the entire contents of each extension reaction into a tube of sodium acetate/ethanol mixture. Mix thoroughly. Vortex the tubes and leave at room temperature for 15 minutes to precipitate the extension products. Precipitation times shorter than 15 minutes will result in the loss of very short extension products. Precipitation times longer than 24 hours will increase the precipitation of unincorporated dye terminators. 4 5 Spin the tubes in a microcentrifuge for 20 min at maximum speed. Carefully aspirate the supernatant with a pipette tip and discard. IMPORTANT The supernatants must be removed completely, as unincorporated dye terminators are dissolved in them. The more residual supernatant left in the tubes, the more unincorporated dye terminators will remain in the samples. 6 7 8 9 Rinse the pellet with 250 L of 70% ethanol. Vortex briey. Spin for 5 minutes in a microcentrifuge at maximum speed. Again, carefully aspirate the supernatant and discard. Dry the pellet in a vacuum centrifuge for 1015 minutes, or until dry. Do not over-dry. (Alternatively, place the tubes with the lids open in a heat block or thermal cycler at 90 C for 1 minute.)

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Sample Electrophoresis
Chapter Summary
Topic

5
See Page 5-2 5-2 5-4 5-6

In This Chapter The following topics are covered in this chapter:

Electrophoresis on the ABI PRISM 3700 DNA Analyzer Electrophoresis on the ABI PRISM 310 Genetic Analyzer Electrophoresis on the ABI PRISM 377 Sequencers Electrophoresis on the ABI PRISM 373 with BigDye Filter Wheel

Sample Electrophoresis 5-1

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Electrophoresis on the ABI PRISM 3700 DNA Analyzer

Overview For information on how to perform sample electrophoresis on the
ABI PRISM 3700 DNA Analyzer, refer to the following manuals: o o

ABI PRISM 3700 DNA Analyzer Sequencing Chemistry Guide (P/N 4309125) ABI PRISM 3700 DNA Analyzer Users Manual (P/N 4306152)

Electrophoresis on the ABI PRISM 310 Genetic Analyzer

Requirements Electrophoresis and data analysis of samples requires the following:
Filter Set E Run Modules
Conguration POP-6 polymer, 1-mL syringe, 61-cm, 50-m i.d. capillary POP-6 polymer, Rapid Sequencing, 1-mL syringe, 47-cm, 50-m i.d. capillary Run Module Seq POP6 (1 mL) E Seq POP6 (1 mL) Rapid E

Dye Set/Primer (Mobility) Files

Instrument ABI PRISM 310, POP-6 polymer ABI PRISM 310, POP-6 polymer, Rapid Sequencing Dye Set/Primer File DT POP6{BD Set-Any Primer} DT POP6{BD Set-Any Primer}

Filter Set E Instrument (Matrix) File Data analysis requires Filter Set E instrument (matrix) le made from the ABI PRISM dRhodamine matrix standards (P/N 4305080). See the Automated DNA Sequencing Chemistry Guide (P/N 4305080, http://www.pebio.com/ab/techsupp/310.html) for more information.

5-2 Sample Electrophoresis

BDT_.book Page 3 Monday, December 13, 1999 1:54 PM

Resuspending the To resuspend the samples: Samples

Step 1 2 3 4 5 Action Resuspend each sample pellet in 1225 L of Template Suppression reagent (TSR, supplied with the polymer). Vortex and spin the samples. Heat the samples at 95 C for 2 minutes, then chill on ice. Vortex and spin the samples again. Place on ice until ready to use. Refer to the ABI PRISM 310 Genetic Analyzer Users Manual (P/N 903565) for guidelines on loading the samples.

Note Although freezing is not recommended on a routine basis, you can keep samples prepared in TSR frozen for several weeks before running on the ABI PRISM 310 Genetic Analyzer with no detectable loss in resolution or base calling.

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Electrophoresis on the ABI PRISM 377 Sequencers

Requirements Electrophoresis and data analysis of samples require the following:
Filter Set E Run Modules
Congurationa 36-cm wtr, 1200 scans/hr, any comb 36-cm wtr, 2400 scans/hr, any comb 48-cm wtr, 1200 scans/hr, any comb DNA Sequencer. Run Module Seq Run 36E-1200 Seq Run 36E-2400 Seq Run 48E-1200

a. Any plate check and prerun modules can be used with the ABI PRISM 377

Dye Set/Primer (Mobility) File: DT {BD Set Any-Primer} The dye set/primer le can be used with 5 and 5.5% Long Ranger gels and 4 and 4.25% polyacrylamide gels (19:1, acrylamide:bis). Filter Set E Instrument (Matrix) File Data analysis requires Filter Set E instrument (matrix) le made from the ABI PRISM dRhodamine matrix standards (P/N 4305080). See the Automated DNA Sequencing Chemistry Guide (P/N 4305080; http://www.pebio.com/ab/techsupp/310.html) for more information.

Using the Lane To resuspend and load samples using the ABI PRISM Lane Guide Lane Guide Kit Identication Kit, refer to the kits protocol (P/N 4313804). Using Long-Read For longer sequencing read lengths follow the gel and buffer Gel and Buffer formulations described in the user bulletin entitled Achieving Longer Formulations High Accuracy Reads on the 377 Sequencer (P/N 4315153).

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Resuspending and Note You can use any plate check and prerun modules. Loading the To resuspend and load the samples: Samples
Step 1 Action Prepare a loading buffer by combining the following in a 5:1 ratio (5 parts deionized formamide to 1 part EDTA with blue dextran): o Deionized formamide o 25 mM EDTA (pH 8.0) with blue dextran (50 mg/mL)

! WARNING ! CHEMICAL HAZARD. Formamide is a

known teratogen (i.e., it can cause birth defects). Wash thoroughly after handling formamide. Wear appropriate protective eyewear, clothing, and gloves. Obtain a copy of the MSDS from the manufacturer. 2 Resuspend each sample pellet in loading buffer as follows: Volume (L): 18- or 36-well 68 2 Volume (L): 48-, 64-, or 96-well 46 1.5

Template PCR product, plasmid, M13 BAC, large DNA 3 4 5

Vortex and spin the samples. Heat the samples at 95 C for 2 minutes to denature. Place on ice until ready to load. Load each sample into a separate lane of the gel as follows: Volume (L): 18- or 36-well 0.751.5 2 Volume (L): 48-, 64-, or 96-well 0.51.0 48-well: 1.5 64-well: 1.5 96-well: 1.01.5

Template PCR product, plasmid, M13 BAC, large DNA

Note If a weak signal is obtained on the ABI PRISM 377 with XL Upgrade, rerun the samples using a CCD gain of 4. Refer to the ABI PRISM 377 DNA Sequencer XL Upgrade Users Manual (P/N 904412) for more information.

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Electrophoresis on the ABI PRISM 373 with BigDye Filter Wheel

Requirements Electrophoresis
Collect BigDye terminator data with Filter Set A on the ABI PRISM 373 sequencer with BigDye Filter Wheel. Data Analysis Data analysis requires a Filter Set A instrument (matrix) le made from the ABI PRISM dRhodamine matrix standards (P/N 4305080) and BigDye terminator mobility le.

Resuspending and To resuspend and load the samples: Loading the Samples Step Action
.

Prepare a loading buffer by combining the following in a 5:1 ratio (5 parts deionized formamide to 1 part EDTA with blue dextran): o Deionized formamide o 25 mM EDTA (pH 8.0) with blue dextran (50 mg/mL)

! WARNING ! CHEMICAL HAZARD. Formamide is a

known teratogen (i.e., it can cause birth defects). Wash thoroughly after handling formamide. Wear appropriate protective eyewear, clothing, and gloves. Obtain a copy of the MSDS from the manufacturer. 2 Resuspend each sample pellet in loading buffer as follows: Volume (L) Template PCR product, plasmid, M13 BAC, large DNA 3 4 18 or 24 well 34 3 32 or 36 well 34 3 48-well 4 2 64-well 4 2

Vortex and spin the samples. Heat the samples at 95 C for 2 minutes to denature. Place on ice until ready to load.

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To resuspend and load the samples: (continued)

Step 5 Action Load each sample into a separate lane of the gel as follows: Volume (L) Template PCR product, plasmid, M13 BAC, large DNA 18 or 24 well 34 3 32 or 36 well 34 3 48-well 24 2 64-well 24 2

Sample Electrophoresis 5-7

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Control DNA Sequence

Control Sequence
TGTAAAACGACGGCCAGT (21 M13 primer) GAATTGTAAT ACGACTCACT ATAGGGCGAA GTACCCGGGG ATCCTCTAGA GTCGACCTGC GCTTGAGTAT TCTATAGTGT CACCTAAATA ATCATGGTCA TAGCTGTTTC CTGTGTGAAA CTCACAATTC CACACAACAT ACGAGCCGGA GTAAAGCCTG GGGTGCCTAA TGAGTGAGCT AATTGCGTTG CGCTCACTGC CCGCTTTCCA CTGTCGTGCC AGCTGCATTA ATGAATCGGC GGAGAGGCGG TTTGCGTATT GGGCGCTCTT GCTCACTGAC TCGCTGCGCT CGGTCGTTCG GCGGTATCAG CTCACTCAAA GGCGGTAATA CAGAATCAGG GGATAACGCA GGAAAGAACA AGGCCAGCAA AAGGCCAGGA ACCGTAAAAA CTGGCGTTTT TCCATAGGCT CCGCCCCCCT ACAAAAATCG ACGCTCAAGT CAGAGGTGGC AGGACTATAA AGATACCAGG CGTTTCCCCC CTCGTGCGCT CTCCTGTTCC GACCCTGCCG ACCTGTCCGC CTTTCTCCCT TCGGGAAGCG TCATAGCTCA CGCTGTAGGT ATCTCAGTTC GTTCGCTCCA AGCTGGGCTG TGTGCACGAA AGCCCGACCG CTGCGCCTTA TCCGGTAACT GTCCAACCCG GTAAGACACG ACTTATCGCC GCCACTGGTA ACAGGATTAG CAGAGCGAGG GTGCTACAGA GTTCTTGAAG TGGTGGCCTA CACTAGAAGG ACAGTATTTG GTATCTGCGC

A
40 80 120 160 200 240 280 320 360 400 440 480 520 560 600 640 680 720 760 800 840 880 920 960 1000

Partial Sequence The pGEM-3Zf(+) sequence below is the the sequence of the 21 M13 of pGEM-3Zf(+) forward primer, followed by the ensuing 1000 bases.
TTCGAGCTCG AGGCATGCAA GCTTGGCGTA TTGTTATCCG AGCATAAAGT AACTCACATT GTCGGGAAAC CAACGCGCGG CCGCTTCCTC GCTGCGGCGA CGGTTATCCA TGTGAGCAAA GGCCGCGTTG GACGAGCATC GAAACCCGAC TGGAAGCTCC CTTACCGGAT TGGCGCTTTC GGTGTAGGTC CCCCCCGTTC ATCGTCTTGA ACTGGCAGCA TATGTAGGCG ACTACGGCTA TCTGCTGAAG

Control DNA Sequence A-1

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Technical Support
Technical Support
To Reach Us on the PE Biosystems web site address is: Web
http://www.pebiosystems.com/techsupport

We strongly encourage you to visit our web site for answers to frequently asked questions, and to learn more about our products. You can also order technical documents and/or an index of available documents and have them faxed or e-mailed to you through our site (see the Documents on Demand section below).

Hours for In the United States and Canada, technical support is available from Telephone 5:30 a.m. to 5:00 p.m. Pacic Time. Technical Support See the back cover of this booklet for how to contact local service
representatives outside of the United States and Canada.

To Reach Us by Call Technical Support at 1-800-831-6844, and select the appropriate option Telephone or Fax (below) for support on the product of your choice at any time during the call. (To in North America open a service call for other support needs, or in case of an emergency, press 1
after dialing 1-800-831-6844.) For Support On This Product ABI Analyzer PRISM 3700 DNA

Dial 1-800-831-6844, and... Press 8 FAX 650-638-5981 FAX 650-638-5981

DNA Synthesis

Press 21

Technical Support B-1

BDT_.book Page 2 Monday, December 13, 1999 1:54 PM

For Support On This Product Fluorescent DNA Sequencing Integrated Thermal Cyclers

Dial 1-800-831-6844, and... Press 22 Press 24 FAX 650-638-5891 FAX 650-638-5891

Documents on Free 24-hour access to PE Biosystems technical documents, including Demand MSDSs, is available by fax or e-mail.
You can access Documents on Demand through the internet or by telephone:
If you want to order... through the internet

Then... Use http://www.pebiosystems.com/techsupport You can search for documents to order using keywords. Up to ve documents can be faxed or e-mailed to you by title.

by phone from the United States or Canada

a. Call 1-800-487-6809 from a touch-tone phone. Have your fax number ready. b. Press 1 to order an index of available documents and have it faxed to you. Each document in the index has an ID number. (Use this as your order number in step d below.) c. Call 1-800-487-6809 from a touch-tone phone a second time. d. Press 2 to order up to ve documents and have them faxed to you.

B-2 Technical Support

BDT_.book Page 3 Monday, December 13, 1999 1:54 PM

If you want to order... by phone from outside the United States or Canada

Then... a. Dial your international access code, then 1-858-712-0317, from a touch-tone phone. Have your complete fax number and country code ready (011 precedes the country code). b. Press 1 to order an index of available documents and have it faxed to you. Each document in the index has an ID number. (Use this as your order number in step d below.) c. Call 1-858-712-0317 from a touch-tone phone a second time. d. Press 2 to order up to ve documents and have them faxed to you.

To Reach Us by Contact technical support by e-mail for help in the following product E-Mail areas.
For this product area Genetic Analysis Protein Sequencing, Peptide and DNA Synthesis Use this e-mail address [email protected] [email protected]

Technical Support B-3

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ABI PRISM DNA Sequencing Kits and Related Products

To order ABI PRISM DNA Sequencing Kits, please contact PE Biosystems at one of the regional sales ofces listed on the back of this protocol. All reagents are quality-controlled in stable formulations. All the kits listed below include protocols. Protocols can also be ordered separately.

dRhodamine Terminator Cycle Sequencing Kits with AmpliTaq DNA Polymerase, FS

P/N 403044 403045 4303143 403041 Kit Ready Reaction Ready Reaction Ready Reaction Protocol Reactions 100 1000 5000

BigDye Terminator Cycle Sequencing Ready Reaction Kits v2.0 with AmpliTaq DNA Polymerase, FS
P/N 4314414 4314415 4314416 4314849 4303237 Kit Ready Reaction Ready Reaction Ready Reaction Ready Reaction Protocol Reactions 100 1000 5000 25,000

BigDye Primer Cycle Sequencing Ready Reaction Kits with AmpliTaq DNA Polymerase, FS
P/N 403051 403049 403052 403050 403057 Primer 21 M13 21 M13 M13 Reverse M13 Reverse Protocol Reactions 100 5000 100 5000

ABI PRISM Lane Guide Lane Identication Kits for use on the 377 Sequencer
P/N 4313682 4313677 4313679 4313804 Kit Lane Guide Lane Guide Lane Guide Protocol Reactions 200 1000 5000

BigDye Terminator Cycle Sequencing Kits with AmpliTaq DNA Polymerase, FS

P/N 4303149 4303150 4303151 4303237 Kit Ready Reaction Ready Reaction Ready Reaction Protocol Reactions 100 1000 5000

ABI PRISM Matrix Standards

P/N 4305609 403047 403047 Kit Matrix Standard Set dRhodamine Matrix Standards dRhodamine Matrix Standards Instrument 3700 310 377/373

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Regional Sales Ofces

THE AMERICAS United States PE Biosystems 850 Lincoln Centre Drive Foster City, CA 94404 Tel: (650) 570-6667 (800) 345-5224 Fax: (650) 572-2743 Canada (Mississauga, Ontario) Tel: (905) 821-8183 (800) 668-6913 Fax: (905) 821-8246 Latin America (Del.A. Obregon, Mexico) Tel: (305) 670-4350 Fax: (305) 670-4349 EUROPE, AFRICA Austria (Wien) Tel: 01 602 3101 Fax: 01 602 5174 Benelux (Nieuwerkerk a/d IJssel, Netherlands) Tel: 31 (0)180-331400 Fax: 31 (0)180-331409 Chekia Rep. (Praha) Tel: 2-61 22 21 64 Fax: 2-61 22 21 68 Denmark (Allerd) Tel: 48 100 400 Fax: 48 100 401 Finland (Espoo) Tel: 09 751 72 700 Fax: 09 751 72 701 France (Paris) Tel: 33-1 69 59 85 85 Fax: 33-1 69 59 85 00 Germany (Weiterstadt) Tel: (0) 6150/101-0 Fax: (0) 6150/101-101 Hungary (Budapest) Tel: 36-1-258-8410 Fax: 36-1-256-9802 Italy (Milano) Tel: (039) 23831 Fax: (039) 2383492 Norway (Oslo) Tel: (0) 22 02 1500 Fax: (0) 22 02 1501 Poland (Warszawa) Tel: (48 22) 866 4010 Fax: (48 22) 866 4020 Portugal (Lisboa) Tel: (351-1) 386 0997 Fax: (351-1) 386 1000 Russia (Moskva) Tel: 095 935 8888 Fax: 095 564 8787 South Africa (Johannesburg) Tel: 27 11 478 0411 Fax: 27 11 478 0349 Spain (Madrid) Tel: (91) 806 1200 Fax: (91) 804 0414 Sweden (Stockholm) Tel: (0)8 619 4400 Fax: (0)8 619 4401 Switzerland (Rotkreuz) Tel: 041-799 7708 Fax: 041-790 0676 United Kingdom (Warrington, Cheshire) Tel: (01925) 825650 Fax: (01925) 282502 All Other European Countries, Middle East, West Asia, Africa Except South Africa Langen, Germany Tel: 49 6103 708 301 Fax: 49 6103 708 310 EASTERN ASIA, CHINA, OCEANIA, PACIFIC RIM Australia (Scoresby, Victoria) Tel: (03) 9212-8585 Fax: (03) 9212-8502 China (Beijing) Tel: 86 10 6238 1156 Fax: 86 10 6238 1162 Hong Kong Tel: 852 2756 6928 Fax: 852 2756 6968 Japan (Chiba) Tel: (0473) 80-8500 Fax: (0473) 80-8505 Korea (Seoul) Tel: 822 592 7238 Fax: 822 532 4908 Malaysia (Kuala Lumpur) Tel: 60 3 758 1118 Fax: 60 3 754 9043 Singapore Tel: 65 896 2118 Fax: 65 896 2147 Taiwan (Taipei Hsisn) Tel: 886 22 698 3505 Fax: 886 22 698 3405 Thailand (Bangkok) Tel: 662 719 6406 Fax: 662 319 9788

P/N 4303237 Rev. D