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DNA Sequencing by Capillary Electrophoresis Chemistry Guide Iii DNA Template Quality

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DNA Sequencing by Capillary Electrophoresis Chemistry Guide Iii DNA Template Quality Chemistry Guide DNA Sequencing by Capillary Electrophoresis Applied Biosystems Chemistry Guide | Second Edition Chemistry Guide DNA Sequencing by Capillary Electrophoresis Applied Biosystems Chemistry Guide Second Edition For Research Use Only. Not for use in diagnostic procedures. Information in this document is subject to change without notice. APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. TO THE FULLEST EXTENT ALLOWED BY LAW, IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF, WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT APPLIED BIOSYSTEMS IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES. NOTICE TO PURCHASER: PLEASE REFER TO THE RESPECTIVE INSTRUMENT'S USER GUIDES, PRODUCT INSERTS, OR APPLIED BIOSYSTEMS PRODUCT PAGE FOR LIMITED LABEL LICENSE OR DISCLAIMER INFORMATION. TRADEMARKS: Applied Biosystems, AB (Design), ABI PRISM, BigDye, BloodPrep, GeneAmp, MicroAmp, MicroSeq, NucPrep, Primer Express, SeqScape, SNaPshot, VariantSEQr, and Veriti are registered trademarks and Hi-Di, KB, methylSEQr, POP, POP-4, POP-5, POP-6, POP-7, SNPbrowser, TargetSeq, Variant Reporter, and XTerminator are trademarks of Applied Biosystems or its affiliates in the US and/or certain other countries. AmpliTaq, AmpliTaq Gold, and TaqMan are registered trademarks of Roche Molecular Systems, Inc. PicoGreen is a registered trademark of Molecular Probes, Inc. All other trademarks are the sole property of their respective owners. © Copyright 2009, Applied Biosystems. All rights reserved. Part Number 4305080 Rev. C 05/2009 Contents Preface How to Use This Guide . vii How to Obtain Support . viii Safety and EMC Compliance Information Safety Conventions Used in This Document . x Chemical Safety . xi Chemical Waste Safety . xi Biological Hazard Safety . xi Chapter 1 Introduction to DNA Sequencing DNA Sequencing Basics . 2 Cycle Sequencing . 5 Capillary Electrophoresis . 9 Data Analysis . 11 Automated DNA Sequencing Workflow . 13 Chapter 2 Applications Overview DNA Sequencing Applications and Approaches . 16 De Novo Sequencing of Genomes . 16 Resequencing . 20 Epigenetics . 23 Microbial Analysis . 27 Chapter 3 DNA Template Preparation Overview . 32 Preparing Vector-Based DNA Templates . 33 Preparing Genomic DNA . 34 Preparing PCR DNA Templates . 37 Primer Design and Quantitation . 38 Purifying PCR Products for Sequencing . 41 DNA Sequencing by Capillary Electrophoresis Chemistry Guide iii DNA Template Quality . 44 DNA Template Quantity . 45 Preparing Templates for Bisulfite Sequencing . 46 Chapter 4 Cycle Sequencing Overview . 54 Choosing a Sequencing Chemistry . 55 Reagent and Equipment Considerations . 61 DNA Quantity . 63 Using DNA Template Controls . 64 Using BigDye Terminators and dGTP BigDye Terminators . 66 Using dRhodamine Terminators . 72 Using BigDye Primers . 73 Direct Sequencing From Cultures . 75 Bisulfite Sequencing . 77 Chapter 5 Purification of Extension Products Overview . 86 Purification with the BigDye® XTerminator™ Purification Kit . 88 Purification by Ethanol Precipitation . 96 Purification with Spin Columns and Spin Plates . 116 Alternative Purification Procedures . 119 Sample Preparation for Electrophoresis . 120 Chapter 6 Capillary Electrophoresis Overview . 128 Calibrating the Instrument . 131 Creating the Plate Record in Data Collection Software . 136 Defining the Results Group . 136 Defining the Instrument Protocol . 137 Defining the Analysis Protocol . 143 Run Parameters for Instrument and Analysis Protocols . 151 Chapter 7 Data Analysis Overview . 158 Reviewing Data with Sequence Scanner Software . 164 Analyzing Data with Sequencing Analysis Software . 170 Analyzing Data with Variant Reporter™ Software . 175 iv DNA Sequencing by Capillary Electrophoresis Chemistry Guide Analyzing Data with SeqScape® Software . 181 Analyzing Data with MicroSeq® ID Analysis Software . 188 Chapter 8 Troubleshooting Troubleshooting Overview . 194 Troubleshooting Workflow . 194 Table of Troubleshooting Symptoms . 201 Troubleshooting Examples . 203 Troubleshooting Index . 255 Appendix A Product Information Peak Color/Base Relationships . 262 Control Sequences . 263 Appendix B Parts List Kits and Reagents . 266 Instrument Parts . 270 Software Products . 278 Miscellaneous Documents . 279 Bibliography Glossary Index DNA Sequencing by Capillary Electrophoresis Chemistry Guide v vi DNA Sequencing by Capillary Electrophoresis Chemistry Guide Preface How to Use This Guide Purpose of This This chemistry guide is designed to familiarize you with Applied Biosystems genetic Guide analyzers for automated DNA sequencing by capillary electrophoresis, to provide useful tips for ensuring that you obtain high quality data, and to help troubleshoot common problems. Chapter 4, “Cycle Sequencing,” Chapter 5, “Purification of.
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