ABI PRISM® 3100 Genetic Analyzer-Sequencing

ABI PRISM 3100 Genetic Analyzer
Sequencing Chemistry Guide
Copyright 2001, Applied Biosystems For Research Use Only. Not for use in diagnostic procedures. FOR LIMITED LICENSE INFORMATION, PLEASE SEE THE ABI PRISM 3100 GENETIC ANALYZER USERS MANUAL.
ABI, BigDye, CATALYST, Hi-Di, and POP-6 are trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries. ABI PRISM, Applied Biosystems and MicroAmp are registered trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries. AmpliTaq, AmpliTaq Gold, and GeneAmp are registered trademarks of Roche Molecular Systems, Inc. Centricon is a registered 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.
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Contents
1 Manual Overview
Chapter Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1 In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1 About This Manual. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2 Purpose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2 Who Should Use This Manual. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2 If You Need More Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2 Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3 Documentation User Attention Words. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3 Chemical Hazard Warning. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3 Chemical Waste Hazard Warning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3 Site Preparation and Safety Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4 Instrument Safety Labels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4 Before Operating the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4 Ordering MSDSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-5
2 About the 3100 Genetic Analyzer

Chapter Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1 In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1 Instrument Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2 3100 Genetic Analyzer Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2 Sequencing Chemistries Supported . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2 Run Cycle Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-3 Capillary Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-4 Capillary vs. Gel Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-4 Advantages of 3100 Capillary Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-5 3100 POP-6 Polymer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-5 Factors Affecting Instrument Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-6 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-6 Template Quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-6 Template Quantity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-8 Compatible Plates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-10 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-10 Compatible Plates from Applied Biosystems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-10
3 BigDye Terminator Chemistry

Chapter Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-1 In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-1
Section: Setting Up BigDye Terminator Reactions for a 96-Well Format . . . . . . . . .3-3

In This Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-3 Preparing the Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-4 Using Control DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-4 Template Quantity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-4 Two Cycle Sequencing Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-5 Preparing 1X Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-5 Preparing 2X Reactions for BACs, PACs, YACs, and Cosmids . . . . . . . . . . . . . . . . . . . 3-6 Preparing 2X Reactions for Bacterial Genomic DNA . . . . . . . . . . . . . . . . . . . . . . . . . . 3-6 Performing Cycle Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-7 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-7 Thermal Cyclers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-7 Modifying the Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-7 Cycle Sequencing Using Standard Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-7 Cycle Sequencing for BACs, PACs, YACs, and Cosmids . . . . . . . . . . . . . . . . . . . . . . . 3-8 Cycle Sequencing for Bacterial Genomic DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-8 Preparing Extension Products for Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-9 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-9 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-9 96-Well Plate Column Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-9 Isopropanol Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-10 Ethanol Precipitation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-11
Section: Setting Up BigDye Terminator Reactions for a 384-Well Format . . . . . . .3-13

In This Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-13 Preparing the Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-14 Using Control DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-14 Template Quantity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-14 Volume Capacity Restrictions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-14 Two Cycle Sequencing Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-15 Preparing Reactions for 384-Well Plate Column Purification . . . . . . . . . . . . . . . . . . . 3-15 Preparing Reactions for Alcohol Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-16 Performing Cycle Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-17 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-17 Thermal Cycler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-17 Modifying the Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-17 Cycle Sequencing Using Standard Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-17 Preparing Extension Products for Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-18 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-18 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-18 384-Well Plate Column Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-18
ii
Ethanol Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-19 Isopropanol Precipitation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-20
4 BigDye Primer Chemistry

Chapter Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-1 In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-1
Section: Setting Up BigDye Primer Reactions for a 96-Well Format . . . . . . . . . . . . 4-3

In This Section. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-3 Preparing the Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-4 Using Control DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-4 Template Quantity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-4 Two Cycle Sequencing Options. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-4 Preparing 1X Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-5 Preparing 2X Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-5 Performing Cycle Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6 Thermal Cyclers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6 Cycle Sequencing Using Standard Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6 Cycle Sequencing for BAC DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-7 Preparing Extension Products for Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-8 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-8 Method. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-8 Ethanol Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-8
Section: Setting Up BigDye Primer Reactions for a 384-Well Format . . . . . . . . . . 4-11

In This Section. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-11 Preparing the Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-12 This Procedure Is Not Recommended . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-12 Using Control DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-12 Template Quantity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-12 Two Cycle Sequencing Options. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-12 Preparing 1X Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-13 Preparing 0.5X Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-13 Performing Cycle Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-14 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-14 Thermal Cycler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-14 Cycle Sequencing Using Standard Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-14 Preparing Extension Products for Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-15 Ethanol Precipitation Not Recommended . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-15
iii
5 dRhodamine Terminator Chemistry

Chapter Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-1 In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-1
Section: Setting Up dRhodamine Terminator Reactions for a 96-Well Format . . . .5-3

In This Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-3 Preparing the Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-4 Using Control DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-4 Template Quantity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-4 Preparing Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-5 Performing Cycle Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-6 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-6 Thermal Cyclers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-6 Cycle Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-6 Preparing Extension Products for Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-7 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-7 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-7 96-Well Reaction Plate Column Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-7 Ethanol/Sodium Acetate Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-8
Section: Setting Up dRhodamine Terminator Reactions for a 384-Well Format . .5-11

In This Section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-11 Preparing the Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-12 Using Control DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-12 Template Quantity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-12 Volume Capacity Restrictions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-12 Preparing Reactions for 384-Well Plate Column Purification . . . . . . . . . . . . . . . . . . . 5-13 Preparing Reactions for Alcohol Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-13 Performing Cycle Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-14 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-14 Thermal Cycler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-14 Cycle Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-14 Preparing Extension Products for Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-15 Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-15 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-15 384-Well Plate Column Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-15 Ethanol/Sodium Acetate Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-15
6 Sample Injections
Chapter Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-1 In This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-1 Preparing Samples for Injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-2 Injection Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-2 Sample Volumes for Reaction Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-2
iv
Covering Sample Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-2 Centrifuging Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-3 For 96 and 384-Well Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-3 Optimizing Electrokinetic Injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-4 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-4 Signal Too Strong . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-4 Signal Too Weak Using 50-cm Array . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-4 Signal Too Weak Using 36-cm Array . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-5 Poor Resolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-5 Optimizing Electrophoresis Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-6 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-6 Run Time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-6 Run Temperature and Run Voltage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-8 Laboratory Temperature and Humidity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-9 For More Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-9 Deionizing and Storing Formamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .A-1 Formamide, Denaturation Agent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .A-1 Option to Purchase or to Make . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .A-1 Purchasing Hi-Di Formamide from Applied Biosystems . . . . . . . . . . . . . . . . . . . . . . . .A-1 The Problem with Commercial Formamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .A-1 Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .A-3 Ion-Exchange Resin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .A-3 Calibrating the Conductivity Meter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .A-4 Preparing EDTA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .A-4 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .A-4 Using the Formamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .A-5 Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-1 Contacting Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-1 Hours for Telephone Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-1 To Contact Technical Support by Telephone or Fax . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-2 To Reach Technical Support Through the Internet . . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-5 To Obtain Documents on Demand . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .B-5 Chemistry Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C-1 Troubleshooting Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C-1
Index
vi
Manual Overview
Chapter Summary
In This Chapter The following topics are covered in this chapter:
Topic About This Manual Safety
1
See Page 1-2 1-3
Manual Overview 1-1
About This Manual

Purpose This manual describes the chemistry protocols for the ABI PRISM 3100 Genetic
Analyzer. It is organized into task-oriented topics, each with a step-by-step description of how to perform the task.
Note This guide is specific to the 3100 Genetic Analyzer. For general chemistry information about kits, dyes, enzymes, protocols, and troubleshooting, please refer to the Automated DNA Sequencing Chemistry Guide (P/N 4305080).
Who Should Use This manual is designed for those persons preparing DNA samples for loading onto This Manual the 3100 Genetic Analyzer. If You Need More Information
If you need more information on... site preparation See the... Part Number 4315835
ABI PRISM 3100 Genetic Analyzer Site Preparation and Safety Guide Automated DNA Sequencing Chemistry Guide ABI PRISM 3100 Genetic Analyzer Quick Start Guide for Sequencing ABI PRISM 3100 Genetic Analyzer Quick Start Guide for Fragment Analysis
general chemistry an abbreviated description of how to sequence on the 3100 Genetic Analyzer an abbreviated description of how to perform fragment analysis on the 3100 Genetic Analyzer
4305080 4315831
4315832
1-2 Manual Overview
Safety
Documentation User Five user attention words appear in the text of all Applied Biosystems user Attention Words documentation. Each word implies a particular level of observation or action as
described below.
Note Calls attention to useful information.
IMPORTANT Indicates information that is necessary for proper instrument operation. ! CAUTION Cautions the user that a potentially hazardous situation could occur, causing injury to the user or damage to the instrument if this information is ignored. ! WARNING Warns the user that serious physical injury or death to the user or other persons could result if these precautions are not taken. ! DANGER Indicates an imminently hazardous situation that, if not avoided, will result in death or serious injury.
Chemical Hazard ! WARNING CHEMICAL HAZARD. Some of the chemicals used with Applied Biosystems Warning instruments and protocols are potentially hazardous and can cause injury, illness, or death.
o Read and understand the material safety data sheets (MSDSs) provided by the chemical manufacturer before you store, handle, or work with any chemicals or hazardous materials. Minimize contact with and inhalation of chemicals. Wear appropriate personal protective equipment when handling chemicals (e.g., safety glasses, gloves, or protective clothing). For additional safety guidelines, consult 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 o
Chemical Waste ! WARNING CHEMICAL WASTE HAZARD. Wastes produced by Applied Biosystems Hazard Warning instruments are potentially hazardous and can cause injury, illness, or death.
o Read and understand the material safety data sheets (MSDSs) provided by the manufacturers of the chemicals in the waste container before you store, handle, or dispose of chemical waste. Handle chemical wastes in a fume hood. Minimize contact with and inhalation of chemical waste. Wear appropriate personal protective equipment when handling chemicals (e.g., safety glasses, gloves, or protective clothing). After emptying the waste container, seal it with the cap provided. Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local, state/provincial, or national environmental and health regulations.
o o
o o
Manual Overview 1-3
Site Preparation and A site preparation and safety guide is a separate document sent to all customers who Safety Guide have purchased an Applied Biosystms instrument. Refer to the guide written for your
instrument for information on site preparation, instrument safety, chemical safety, and waste profiles.
Instrument Safety Safety labels are located on the instrument. Each safety label has three parts: Labels o A signal word panel, which implies a particular level of observation or action (e.g.,
CAUTION or WARNING). If a safety label encompasses multiple hazards, the signal word corresponding to the greatest hazard is used.
o o
A message panel, which explains the hazard and any user action required. A safety alert symbol, which indicates a potential personal safety hazard. See the ABI PRISM 3100 Genetic Analyzer Site Preparation and Safety Guide for an explanation of all the safety alert symbols provided in several languages.
Before Operating the Ensure that everyone involved with the operation of the instrument has: Instrument o Received instruction in general safety practices for laboratories
o o Received instruction in specific safety practices for the instrument Read and understood all related MSDSs
! CAUTION Avoid using this instrument in a manner not specified by Applied Biosystems. Although the instrument has been designed to protect the user, this protection can be impaired if the instrument is used improperly.
1-4 Manual Overview
Ordering MSDSs You can order free additional copies of MSDSs for chemicals manufactured or
distributed by Applied Biosystems using the contact information below.
To order MSDSs... Over the Internet Then... a. b. c. d. e. Go to our Web site at www.appliedbiosystems.com/techsupport. Click MSDSs. Enter keywords (or partial words), or a part number, or the MSDSs Documents on Demand index number. Click Search. Click 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 By telephone from Canada
Use Documents on Demand on B-5. Dial 1-800-327-3002, then press 1.
To order 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 Regional Offices Sales and Service on B-4.
For chemicals not manufactured or distributed by Applied Biosystems, call the chemical manufacturer.
Manual Overview 1-5
About the 3100 Genetic Analyzer 2

Chapter Summary
Topic Instrument Overview Capillary Electrophoresis Factors Affecting Instrument Performance Compatible Plates
2
See Page 2-2 2-4 2-6 2-10
About the 3100 Genetic Analyzer 2-1
Instrument Overview
3100 Genetic The ABI PRISM 3100 Genetic Analyzer is an automated, high-throughput, capillary Analyzer electrophoresis system used for analyzing fluorescently labeled DNA fragments. Description Sequencing The following chemistries are currently supported for use with the 3100 Genetic Chemistries Analyzer: Supported o ABI PRISM BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit v2.0
25,000 reactions (P/N 4314849) 5000 reactions (P/N 4314416) 1000 reactions (P/N 4314415) 100 reactions (P/N 4314414) o ABI PRISM BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit 5000 reactions (P/N 4303151) 1000 reactions (P/N 4303150) 100 reactions (P/N 4303149) o o ABI PRISM dGTP BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit 100 reactions (P/N 4307175) ABI PRISM BigDyeTM Primer Cycle Sequencing Ready Reaction Kit 5000 reactions with 21 M13 primer (P/N 403049) 5000 reactions with M13 reverse primer (P/N 403050) 100 reactions with 21 M13 primer (P/N 403051) 100 reactions with M13 reverse primer (P/N 403052) o ABI PRISM dRhodamine Terminator Cycle Sequencing Ready Reaction Kit 5000 reactions (P/N 4303143) 1000 reactions (P/N 403045) 100 reactions (P/N 403044)
2-2 About the 3100 Genetic Analyzer
Run Cycle Overview
Step 1 2 3 4
Event DNA samples are prepared for sequencing in 96- or 384-well plates and placed on the autosampler of the instrument. The capillaries are filled with 3100 POP-6 polymer, a medium that separates the DNA fragments. The autosampler positions the 16 capillaries into the sample wells. The fluorescently labeled DNA is loaded into the capillary by a short period of electrophoresis called electrokinetic injection. The capillaries are rinsed with water to remove sample adhering to the capillary sides. The autosampler moves and positions the 16 capillaries into the buffer chamber for electrophoresis. When the DNA fragments reach the detection window, the laser beam excites the dye molecules and causes them to fluoresce. The fluorescence emissions from 16 samples are collected simultaneously and spectrally separated by a reflective spectrograph. The fluorescence emissions are focused as columns of light onto the charge-coupled device (CCD) that is part of the CCD camera. The 3100 sequencing Data Collection software reads and interprets the fluorescence data, then displays the data as an electropherogram.
5 6 7
Capillary Electrophoresis
Capillary vs. Gel Main Differences Electrophoresis Capillary and gel electrophoresis both separate DNA fragments by size through a
sieving matrix in an electric field. There are three main differences between these separation methods: o o o The structure of the sieving component The dissipation of heat The method of sample loading
The Sieving Component Slab-gel electrophoresis, performed on the ABI PRISM 373 and 377 DNA Sequencers, uses a gel created by the crosslinking of polyacrylamide and bis-acrylamide. The degree of crosslinking affects the porosity of the gel and thus the potential resolution of the DNA fragments. Once the gel matrix polymerizes, the gel is in a solid stationary phase. Capillary electrophoresis is performed on the 3100 Genetic Analyzer, 3700 DNA Analyzer and the ABI PRISM 310 Genetic Analyzer. The medium for capillary electrophoresis is a linear flowable polymer called 3100 POP-6 polymer. These polymers can be pumped into and out of the capillaries for each new run. Heat Dissipation The method of heat dissipation for slab gel electrophoresis differs from that of capillary electrophoresis. During capillary electrophoresis heat is evenly and efficiently dissipated because there is more surface area in a capillary than in a gel plate. This enables higher voltages to be applied to migrating extension fragments without loss of resolution. Sample Loading Slab gels are loaded by inserting a pipet tip between two glass plates and into the pre-formed wells of the gel slab. The sample (including salts and proteins) is layered onto the top of the gel slab. Samples for capillary electrophoresis are loaded into the capillary via electrokinetic injection. During electrokinetic injection negative ions such as extension fragments are injected into the capillary. However, no sample volume is injected or lost.
Advantages of 3100 The advantages of capillary electrophoresis on the 3100 Genetic Analyzer are: Capillary o Rapid separation of DNA fragments. Electrophoresis
o o o o
No gel pouring required. The 3100 Genetic Analyzer uses a liquid polymer, which is flowable and replenishable. No manual sample loading required. No gel tracking required. Increased sensitivity resulting from the simultaneous detection of 16 samples.
3100 POP-6 Polymer The 3100 POP-6 polymer (P/N 4316357) is a flowable polymer that is available for use
on the 3100 Genetic Analyzer.
Factors Affecting Instrument Performance

Overview The quality and quantity of DNA in a reaction can affect the performance of the 3100
Genetic Analyzer. Template Quality The presence of residual salts, proteins, RNA, and detergents can interfere with capillary electrophoresis and electrokinetic injection. Your current template purification methods may have to be modified to remove residual salts, proteins, and detergents. Template Quantity The amount of DNA template used in a sequencing reaction can affect the quality of data. Excess template makes data appear top heavy with strong peaks at the beginning of the run that fade rapidly. Too little template or primer reduces the signal strength and peak height. In the worst case, the noise level increases so that bases cannot be called.
Template Quality When preparing DNA templates, it is critical to avoid the following:
o o o o Residual salts Proteins Residual detergents Residual RNA
Effect of Residual Salts The 3100 Genetic Analyzer is especially susceptible to salt in samples, either from template preparation or from cycle sequencing reactions. The negative ions in salts are preferentially injected into the capillaries during electrokinetic injection, leading to lower signal. Applied Biosystems scientists recommend implementing an efficient method to remove excess salts. Refer to the Automated DNA Sequencing Chemistry Guide for protocols. Figure 2-1 shows the effect of salt during electrokinetic injection. The capillary marked A is a standard prepared using the recommended protocol; the capillaries marked B are standards with 10 mM NaCl added. The gel image in Figure 2-1 was artificially created by compressing the real-time data.
IMPORTANT When excess salts are present in the sample, negative ions compete and interfere with the injection of larger DNA extension fragments. This leads to shortened read lengths.
Figure 2-1
The effect of salt during electrokinetic injection
Effect of Proteins Many DNA preparation methods for sequencing require the recovery of DNA from lysed bacterial cultures. Unless DNA is carefully purified, protein can remain in the DNA samples. Protein can be injected and adhere to the walls of the capillary, thus affecting data resolution and capillary lifetime. Effect of Residual Detergents Some methods of template preparation, such as the Thermomax method for M13 preparation, use detergents such as Triton X-100 to lyse the protein coat of phage particles. Other detergents, such as sodium dodecyl sulfate (SDS), are used in plasmid purification protocols to lyse bacterial cells. Small, negatively charged detergents may be preferentially injected over DNA during electrokinetic injection. If present at high levels, detergents such as Triton X-100 and SDS will impact the life of the capillary and the quality of the sequencing data. Effect of Residual RNA Residual RNA that is present in DNA template preps competes with the DNA for injection into the capillary array. Residual RNA has the same effect as excess salt, that is, decreased signal and shortened read lengths.
Template Quantity Excess Template in Your Sample

Loading too much template can potentially interfere with capillary flow, or shorten the life of capillaries. When DNA template is present in excess, it will behave as proteins would, and accumulate in the capillary. This affects data resolution. Figure 2-2 on page 2-8 shows raw data from a BigDye terminator reaction containing excess template. The presence of excess template results in top heavy data and short reads. Electrokinetic injection enhances the effects of excess template by preferentially injecting shorter DNA fragments.
Figure 2-2
Raw data showing the effect of excess template in a BigDye terminator reaction
While Figure 2-2 shows raw data from a BigDye terminator reaction containing excess template, Figure 2-3 on page 2-9 shows analyzed data for this sample. The electropherogram in Figure 2-3 displays peaks that are clearly off-scale and have overall shortened read lengths. The presence of excess template in the reaction and the preferential electrokinetic injection of small DNA fragments cause this effect.
Figure 2-3
Electropherogram from the sample run in Figure 2-2
Compatible Plates
Introduction Samples must either be prepared in a plate or transferred to a plate before being
placed on the autosampler. Make sure that the plate is securely placed on the autosampler.
Compatible Plates Applied Biosystems supplies two types of plates (shown below) that are compatible from Applied with the 3100 Genetic Analyzer. Biosystems IMPORTANT Presently, these are the plates recommended for use on the 3100 Genetic
Analyzer. Other plate types may be of different dimensions and thus affect instrument performance. Plates of varying tube depths may damage the array or the autosampler.
MicroAmp Optical 96-Well Reaction Plate
MicroAmp 384-Well Reaction Plate
BigDye Terminator Chemistry

Chapter Summary
Topic
3
See Page 3-3 3-4 3-7 3-9 3-13 3-14 3-17 3-18
Setting Up BigDye Terminator Reactions for a 96-Well Format Preparing the Reactions Performing Cycle Sequencing Preparing Extension Products for Electrophoresis Setting Up BigDye Terminator Reactions for a 384-Well Format Preparing the Reactions Performing Cycle Sequencing Preparing Extension Products for Electrophoresis
BigDye Terminator Chemistry 3-1
3-2 BigDye Terminator Chemistry
Section: Setting Up BigDye Terminator Reactions for a 96-Well Format

In This Section This protocol describes how to prepare BigDye terminator cycle sequencing
reactions in MicroAmp Optical 96-Well Reaction Plates.
Topic Setting Up BigDye Terminator Reactions for a 96-Well Format Preparing the Reactions Performing Cycle Sequencing Preparing Extension Products for Electrophoresis See Page 3-3 3-4 3-7 3-9
Preparing the Reactions

Using Control DNA It is strongly recommended that you include a control DNA template as one of the
templates in a set of sequencing reactions. The results from the control can help determine whether failed reactions are the result of poor template quality or sequencing reaction failure. We recommend M13mp18 as a single-stranded control and pGEM-3Zf(+) as a double-stranded control. All Applied Biosystems DNA sequencing kits provide pGEM control DNA. All dye terminator cycle sequencing kits include a 21 M13 control primer.
Template Quantity The table below shows the template quantities for the BigDye terminator chemistry for
a 1X cycle-sequencing run.
Template PCR product: 100200 bp 200500 bp 5001000 bp 10002000 bp >2000 bp Single-stranded Double-stranded Cosmid, BAC Bacterial genomic DNA 13 ng 310 ng 520 ng 1040 ng 40100 ng 50100 ng 200500 ng 0.51.0 g 23 g Template Quantity
Two Cycle The flexibility of the BigDye terminator chemistry allows two options for cycle Sequencing Options sequencing, as shown in the table below.
Reaction Type 1X Template o PCR product o Plasmid o M13 High-sensitivity (2X) o Large DNA templates o Bacterial genomic DNA Modified Preparing 2X Reactions for BACs, PACs, YACs, and Cosmids on page 3-6. or Preparing 2X Reactions for BACs, PACs, YACs, and Cosmids on page 3-6. Cycle Standard See... Preparing 1X Reactions on page 3-5.
Preparing 1X To prepare 1X BigDye terminator reactions: Reactions

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 Quantity 8.0 L 50100 ng 200500 ng 1100 ng (depending on size, see Template Quantity on page 3-4). 3.2 pmol q.s. 20 L
Primer Deionized water Total volume 2 3 Mix well and spin briefly.
Continue with Performing Cycle Sequencing on page 3-7.
Preparing 2X To prepare high-sensitivity (2X) BigDye terminator reactions for BACs, PACs, YACs, Reactions for BACs, and cosmids: PACs, YACs, and Action Cosmids Step
1 For each reaction, add the following reagents to a separate tube: Reagent Terminator Ready Reaction Mix DNA Template Primer Deionized water Total volume 2 3 Mix well and spin briefly. Continue with Performing Cycle Sequencing on page 3-7. Quantity 16 L 0.51.0 g 510 pmol q.s. 40 L
Preparing 2X To prepare high-sensitivity (2X) BigDye terminator reactions for bacterial genomic Reactions for DNA: Bacterial Genomic Action DNA Step
1 For each reaction, add the following reagents to a separate tube: Reagent Terminator Ready Reaction Mix Templatea Primer Deionized water Total Volume 2 3 Mix well and spin briefly. Continue with Performing Cycle Sequencing on page 3-7. Quantity 16 L 23 g 613 pmol q.s. 40 L
a. Shearing the DNA by passing it several times through a 21-guage, 1-inch long needle can improve signals.
Performing Cycle Sequencing

Overview The following three protocols are used for BigDye terminator cycle sequencing
reactions. o o o Cycle Sequencing Using Standard Conditions Cycle Sequencing for BACs, PACs, YACs, and Cosmids Cycle Sequencing for Bacterial Genomic DNA
Thermal Cyclers These protocols have been optimized for all Applied Biosystems thermal cyclers:
o o o The ABI PRISM CATALYST 800 Molecular Biology Lab Station The ABI PRISM 877 Integrated Thermal Cycler The GeneAmp PCR Systems 9600 and 9700 (in 9600 Emulation Mode).
IMPORTANT The protocols contained in this section should work for all of these instruments. If you use a thermal cycler not manufactured by Applied Biosystems, you may need to optimize thermal cycling conditions. Ramping time is very important. If the thermal ramping time is too fast (>1 C/second), poor (noisy) data may result.
Modifying the These protocols work for a variety of templates. However, the following modifications Protocols may be made:
o o o o For short PCR products, a reduced numbers of cycles can be used (e.g., 20 cycles for a 300-bp or smaller fragment). If the Tm of a primer is >60 C, the annealing step can be eliminated. If the Tm of a primer is <50 C, increase the annealing time to 30 seconds or decrease the annealing temperature to 48 C. For templates with high GC content (>70%), heat the tubes at 98 C for 5 minutes before cycling to help denature the template.
Cycle Sequencing To perform cycle sequencing under standard conditions: Using Standard Step Action Conditions
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. Continue with Preparing Extension Products for Electrophoresis on page 3-9.
a. Rapid thermal ramp is 1 C/second.
Cycle Sequencing for To perform cycle sequencing for BACs, PACs, YACs, and cosmids: BACs, PACs, YACs, Step Action and Cosmids
1 2 3 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 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. Continue with Preparing Extension Products for Electrophoresis on page 3-9.
a. Increasing the number of cycles in step 3 increases signal. b. Rapid thermal ramp is 1 C/second.
Cycle Sequencing for To perform cycle sequencing for bacterial genomic DNA: Bacterial Genomic Step Action DNA
1 2 3 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: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 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. Continue with Preparing Extension Products for Electrophoresis on page 3-9.
a. Increasing the number of cycles in step 3 increases signal. b. Rapid thermal ramp is 1 C/second.
Preparing Extension Products for Electrophoresis

Overview 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 basecalling. For an example of the effect of unincorporated dye terminators on sequencing data, refer to the Automated DNA Sequencing Chemistry Guide.
Methods Several methods for preparing extension products for electrophoresis are presented to
offer a choice of reagents and processes. We recommend performing controlled reactions with each method to determine the one that works best for you. o The spin column and 96-well reaction plate methods remove most or all excess terminators if performed correctly, but are more costly than precipitation methods.
IMPORTANT Precipitation methods are cheaper, but are more likely to leave unincorporated dye-labeled terminators that can obscure data at the beginning of the sequence. We recommend that you use anhydrous (100%) isopropanol or nondenatured ethanol. Refer to Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions User Bulletin (P/N 4304665). IMPORTANT When using alcohol precipitation methods, a 70% isopropanol or 70% ethanol wash step is required. This removes residual salts and residual unincorporated dyes. If salts and unincorporated dyes are not removed from the sequencing reaction, they will compete with the extension fragments during electrokinetic injection and result in weak signals.
For BigDye terminator chemistries, the following methods are recommended:

Chemistry BigDye Terminator Recommended Methods Purifying PCR Fragments by Ultrafiltration See
Automated DNA Sequencing Chemistry Guide

3-9 3-10 3-11
96-Well Plate Column Purification Isopropanol Precipitation Ethanol Precipitation
96-Well Plate For large-scale procedures, you can use the following commercially available 96-well Column Purification purification plates:
o o o o 96-Well Spin Columns, Gel Filtration Kit (Edge Biosystems, P/N 94880) 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)
Refer to the manufacturers instructions procedures.
Isopropanol You will need the following reagents and equipment for this procedure: Precipitation o Variable speed table-top centrifuge with microtiter plate, capable of reaching at
least 1400 g o MicroAmp strip caps or adhesive-backed aluminum foil tape (3M Scotch Tape 431 or 439)1
IMPORTANT 75% Isopropanol (2-propanol) or 100% isopropanol (anhydrous) at room temperature Note This procedure does not use salt.
To perform isopropanol precipitation:

Step 1 2 Action Remove the 96-well reaction plate from the thermal cycler. Remove the caps. Add one of the following: o 80 L of 75% isopropanol -oro 20 L of deionized water and 60 L of 100% isopropanol The final isopropanol concentration should be 60 5%. ! WARNING CHEMICAL HAZARD. Isopropanol is a flammable liquid and vapor. It may cause eye, skin, and upper respiratory tract irritation. Prolonged or repeated contact may dry skin and cause irritation. It may cause central nervous system effects such as drowsiness, dizziness, and headache, etc. Please read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. 3 Seal the plate with strip caps or by applying a piece of 3M Scotch Tape 431 or 439 adhesive-backed aluminum foil tape. Press the foil onto the plate to prevent any leakage. Invert the plate a few times to mix, or vortex. Leave the plate at room temperature for 15 minutes to precipitate the extension products. Note Precipitation times <15 minutes will result in the loss of very short extension products. Precipitation times >24 hours will increase the precipitation of unincorporated dye terminators. 6 Place the plate in a table-top centrifuge with plate 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 IMPORTANT Proceed to the next step immediately. If not possible, then spin the plate for an additional 2 minutes immediately before performing the next step. 7 Without disturbing the precipitates, remove the adhesive tape and discard the supernatant by inverting the plate onto a paper towel. Remove as much supernatant as possible. Rinse the pellet by adding 150 L of 70% isopropanol to each well. Seal the plate with adhesive tape and invert the plate a few times to mix.
4 5
8 9
1.
In the USA, contact 3M at (800) 364-3577 for your local 3M representative. Use of other tapes may result in leakage or contamination of the sample.
To perform isopropanol precipitation: (continued)

Step 10 11 12 13 Action Place the plate in a table-top centrifuge and spin at 2000 g for 10 minutes. Remove the adhesive tape. Discard the wash onto a paper towel that is folded to the size of the plate. Place the inverted plate with the towel into the table-top centrifuge and spin at 700 g for 1 minute. Remove the plate and discard the paper towel. Note Pellets may or may not be visible. Vacuum drying of the samples is not necessary.
Ethanol With ethanol precipitation, traces of unincorporated terminators may be seen at the Precipitation 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.
Where 95% ethanol is recommended in precipitation protocols, purchase IMPORTANT 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 final concentrations of ethanol, which can affect some protocols.
You will need the following equipment and reagents for this procedure: o o Variable speed table-top centrifuge with microtiter plate tray, capable of reaching at least 1400 g MicroAmp strip caps or adhesive-backed aluminum foil tape (3M Scotch Tape 431 or 439)2
IMPORTANT 95% Ethanol (ACS reagent grade), non-denatured Note This procedure does not use salt.
To perform ethanol precipitation:

Step 1 2 Action Remove the 96-well reaction plates from the thermal cycler. Remove the caps from each plate. Add the following: o 16 L of deionized water o 64 L of non-denatured 95% ethanol (EtOH) The final ethanol concentration should be 60 3%. ! WARNING CHEMICAL HAZARD. Ethanol is a flammable liquid and vapor. It may cause eye, skin, and upper respiratory tract irritation. Prolonged or repeated contact may dry skin. Exposure may cause central nervous system depression and liver damage. Please read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. 3 Seal the plate with strip caps or by applying a piece of 3M Scotch Tape 431 or 439 adhesive-backed aluminum foil tape. Press the foil onto the plate to prevent any leakage.
2.
In the USA contact 3M at (800) 364-3577 for your local 3M representative. Use of other tapes may result in leakage or contamination of the sample.
To perform ethanol precipitation: (continued)

Step 4 5 Action Invert the plate a few times to mix. Leave the plate at room temperature for 15 minutes to precipitate the extension products. Note Precipitation times <15 minutes will result in the loss of very short extension products. Precipitation times >24 hours will increase the precipitation of unincorporated dye terminators. 6 Place the plate in a table-top centrifuge with plate adaptor and spin it at the maximum speed, which must be 1400 g but <3000 g: o 14002000 g for 45 minutes o 20003000 g for 30 minutes IMPORTANT Proceed to the next step immediately. If not possible, then spin the plate 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 plate onto a paper towel. Remove as much supernatant as possible. Rinse the pellet by adding 150 L of 70% ethanol to each well. Seal the plates with adhesive tape and invert the plate a few times to mix. Place the plate in a table-top centrifuge and spin at 2000 g for 10 minutes. Remove the adhesive tape and discard the wash onto a paper towel that is folded to the size of the plate. Place the inverted plate with the towel into the table-top centrifuge and spin at 700 g for 1 minute. Remove the plate 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
Section: Setting Up BigDye Terminator Reactions for a 384-Well Format

In This Section This section describes how to prepare BigDye terminator cycle sequencing
Topic Setting Up BigDye Terminator Reactions for a 384-Well Format Preparing the Reactions Performing Cycle Sequencing Preparing Extension Products for Electrophoresis See Page 3-13 3-14 3-17 3-18

Template Quantity The table below shows the template quantities for the BigDye terminator chemistry.
Template PCR product: 100200 bp 200500 bp 5001000 bp 10002000 bp >2000 bp Single-stranded Double-stranded Cosmid, BAC Bacterial genomic DNA 13 ng 310 ng 520 ng 1040 ng 40100 ng 50100 ng 200500 ng Not recommended Not recommended Template Quantity
Volume Capacity IMPORTANT The wells in the 384-well plates have a maximum capacity of 35 L. A portion of Restrictions this capacity will be utilized during the post-reaction clean up step. For this reason, choose your
reaction setup based on the method you will use to prepare extension products for electrophoresis. If you are purifying extension products using... 384-well plate column purification Then set up your reactions following this procedure ... o Preparing Reactions for 384-Well Plate Column Purification on page 3-15. o Preparing Reactions for Alcohol Precipitation on page 3-16. o ethanol precipitation o isopropanol precipitation Preparing Reactions for Alcohol Precipitation on page 3-16a.
a. This protocol ensures that you will not exceed the volume capacity of the 384-well plate.
Two Cycle The flexibility of the BigDye terminator chemistry allows two options for cycle Sequencing Options sequencing, as shown in the table below.
Post-Reaction Purification Method 384-Well Plate Column Purification o 384-well plate column purification o Ethanol precipitation o Isopropanol precipitation
Template o PCR product o Plasmid o M13 o PCR product o Plasmid o M13
Cycle Standard
See... Preparing Reactions for 384-Well Plate Column Purification on page 3-15. Preparing Reactions for Alcohol Precipitation on page 3-16.
Standard
Preparing Reactions IMPORTANT Follow this reaction set up if extension products will be purified using 384-well for 384-Well Plate plate column purification. If you are purifying extension products using alcohol precipitation, Column Purification refer to Preparing Reactions for Alcohol Precipitation on page 3-16.
To prepare BigDye terminator reactions for 384-well plate column purification:
Preparing Reactions Follow this procedure if you are preparing BigDye terminator reactions and preparing for Alcohol the extension products for electrophoresis using any of the methods: Precipitation o 384-well plate column purification
o o Ethanol precipitation Isopropanol precipitation

Overview The following protocol is used for BigDye terminator cycle sequencing reactions.
o Cycle Sequencing Using Standard Conditions
Thermal Cycler These protocols have been optimized for the GeneAmp PCR System 9700 (in 9600
Emulation Mode).
IMPORTANT If you use a thermal cycler not manufactured by Applied Biosystems, you may need to optimize thermal cycling conditions. Ramping time is very important. If the thermal ramping time is too fast (>1 C/second), poor (noisy) data may result.
Modifying the These protocols work for a variety of templates. However, the following modifications Protocols may be made:
o o o o For short PCR products, a reduced numbers of cycles can be used (e.g., 20 cycles for a 300-bp or smaller fragment). If the Tm of a primer is >60 C, the annealing step can be eliminated. If the Tm of a primer is <50 C, increase the annealing time to 30 seconds or decrease the annealing temperature to 48 C. For templates with high GC content (>70%), heat the tubes at 98 C for 5 minutes before cycling to help denature the template.
Cycle Sequencing To perform cycle sequencing using standard conditions: Using Standard Step Action Conditions
1 2 Place the tubes in a thermal cycler and set the volume to 20 L for 1X reactions or 10 L for half-volume reactions. 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. Continue with Preparing Extension Products for Electrophoresis on page 3-18.

Methods Several methods for preparing extension products for electrophoresis are presented to
offer a choice of reagents and processes. We recommend performing controlled reactions with each method to determine the one that works best for you. o The spin column and 384-well reaction plate methods remove most or all excess terminators if performed correctly, but are more costly than precipitation methods.
IMPORTANT Precipitation methods are cheaper, but are more likely to leave unincorporated dye-labeled terminators that can obscure data at the beginning of the sequence. We recommend that you use anhydrous (100%) isopropanol or nondenatured ethanol. Refer to Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions User Bulletin (P/N 4304665). IMPORTANT When using precipitation methods, a 70% isopropanol or 70% ethanol wash step is required. This removes residual salts and residual unincorporated dyes. If salts and unincorporated dyes are not removed from the sequencing reaction, they will compete with the extension fragments during electrokinetic injection and result in weak signals.
For BigDye terminator chemistries, the following methods are recommended:

Chemistry BigDye Terminator Recommended Methods Purifying PCR Fragments by Ultrafiltration See
Automated DNA Sequencing Chemistry Guide

3-18 3-19 3-20
384-Well Plate Column Purification Ethanol Precipitation Isopropanol Precipitation
384-Well Plate For large-scale procedures, you can use the following commercially available 384-well Column Purification reaction plate:
o o ArrayIt (Telechem, P/N DTC-384-100) 384 System I (Edge Biosystems, P/N 95674)
Refer to the manufacturers instructions for procedures.
Ethanol With ethanol precipitation, traces of unincorporated terminators may be seen at the Precipitation 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.
Where 95% ethanol is recommended in precipitation protocols, purchase IMPORTANT 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 final concentrations of ethanol, which can affect some protocols.
You will need the following equipment and reagents for this procedure: o o o Variable speed table-top centrifuge with microtiter plate tray, capable of reaching at least 1400 g Adhesive-backed aluminum foil tape (3M Scotch Tape 431 or 439)3 95% Ethanol (ACS reagent grade), non-denatured
This procedure does not use salt.
Note

Step 1 2 Action Remove the 384-well reaction plates from the thermal cycler. Remove the seal from each plate. To the 10 L reaction volume add the following: o 18 L of non-denatured 95% ethanol (EtOH) The final ethanol concentration should be 60 3%. ! WARNING CHEMICAL HAZARD. Ethanol is a flammable liquid and vapor. It may cause eye, skin, and upper respiratory tract irritation. Prolonged or repeated contact may dry skin. Exposure may cause central nervous system depression and liver damage. Please read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. 3 4 5 Seal the plate with a piece of 3M Scotch Tape 431 or 439 adhesive-backed aluminum foil tape. Press the foil onto the plate to prevent any leakage. Invert the plate a few times to mix. Leave the plate at room temperature for 15 minutes to precipitate the extension products. Note Precipitation times <15 minutes will result in the loss of very short extension products. Precipitation times >24 hours will increase the precipitation of unincorporated dye terminators. 6 Place the plate in a table-top centrifuge with plate adaptor and spin it at the maximum speed, which must be 1400 g but <3000 g: o 14002000 g for 45 minutes o 20003000 g for 30 minutes IMPORTANT Proceed to the next step immediately. If not possible, then spin the plate for 2 minutes more immediately before performing the next step.
3.

Step 7 Action Without disturbing the precipitates, remove the adhesive tape and discard the supernatant by inverting the plate onto a paper towel. Remove as much supernatant as possible. Rinse the pellet by adding 35 L of 70% ethanol to each well. Seal the plates with adhesive tape and invert the plate a few times to mix. Place the plate in a table-top centrifuge and spin at 2000 g for 10 minutes. Remove the adhesive tape and discard the wash onto a paper towel that is folded to the size of the plate. Place the inverted plate with the towel into the table-top centrifuge and spin at 700 g for 1 minute. Remove the plate 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
Isopropanol You will need the following reagents and equipment for this procedure: Precipitation o Variable speed table-top centrifuge with microtiter plate, capable of reaching at
least 1400 g o o Adhesive-backed aluminum foil tape (3M Scotch Tape 431 or 439)4 100% isopropanol (anhydrous) at room temperature
Note
To perform isopropanol precipitation:

Step 1 2 Action Remove the 384-well reaction plates from the thermal cycler. Remove the seal from each plate. To the 10 L reaction volume add the following: o 15 L of 100% isopropanol The final isopropanol concentration should be 60 5%. ! WARNING CHEMICAL HAZARD. Isopropanol is a flammable liquid and vapor. It may cause eye, skin, and upper respiratory tract irritation. Prolonged or repeated contact may dry skin and cause irritation. It may cause central nervous system effects such as drowsiness, dizziness, and headache, etc. Please read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. 3 4 Seal the plate by applying a piece of 3M Scotch Tape 431 or 439 adhesive-backed aluminum foil tape. Press the foil onto the plate to prevent any leakage. Invert the plate a few times to mix, or vortex.
4.
In the USA, contact 3M at (800) 364-3577 for your local 3M representative. Use of other tapes may result in leakage or contamination of the sample.
To perform isopropanol precipitation: (continued)

Step 5 Action Leave the plate at room temperature for 15 minutes to precipitate the extension products. Note Precipitation times <15 minutes will result in the loss of very short extension products. Precipitation times >24 hours will increase the precipitation of unincorporated dye terminators. 6 Place the plate in a table-top centrifuge with plate 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 IMPORTANT Proceed to the next step immediately. If not possible, then spin the plate for an additional 2 minutes immediately before performing the next step. 7 Without disturbing the precipitates, remove the adhesive tape and discard the supernatant by inverting the plate onto a paper towel. Remove as much supernatant as possible. Rinse the pellet by adding 35 L of 70% isopropanol to each well. Seal the plate with adhesive tape and invert the plate a few times to mix. Place the plate in a table-top centrifuge and spin at 2000 g for 10 minutes. Remove the adhesive tape. Discard the wash onto a paper towel that is folded to the size of the plate. Place the inverted plate with the towel into the table-top centrifuge and spin at 700 g for 1 minute. Remove the plate 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
BigDye Primer Chemistry

Chapter Summary
Topic Setting Up BigDye Primer Reactions for a 96-Well Format Preparing the Reactions Performing Cycle Sequencing Preparing Extension Products for Electrophoresis Setting Up BigDye Primer Reactions for a 384-Well Format Preparing the Reactions Performing Cycle Sequencing Preparing Extension Products for Electrophoresis
4
See Page 4-3 4-4 4-6 4-8 4-11 4-12 4-14 4-15
BigDye Primer Chemistry 4-1
4-2 BigDye Primer Chemistry
Section: Setting Up BigDye Primer Reactions for a 96-Well Format

In This Section This protocol describes how to prepare BigDye primer cycle sequencing reactions in
MicroAmp Optical 96-Well Reaction Plates.
Topic Setting Up BigDye Primer Reactions for a 96-Well Format Preparing the Reactions Performing Cycle Sequencing Preparing Extension Products for Electrophoresis See Page 4-3 4-4 4-6 4-8

Template Quantity The table below shows recommended template quantities for the BigDye primer
chemistry.
Template PCR product: 100200 bp 200500 bp 5001000 bp 10002000 bp >2000 bp Single-stranded Double-stranded Cosmid, BAC Bacterial genomic DNA 25 ng 510 ng 1020 ng 2050 ng 50150 ng 150400 ng 200800 ng 0.51.0 g Not recommended Template Quantity
Two Cycle The flexibility of the BigDye primer chemistry allows two options for cycle sequencing, Sequencing Options as shown in the table below.
Reaction Type 1X Template o PCR product o Plasmid o M13 High-sensitivity (2X) o Large DNA template containing 21 M13 and/or M13 Reverse priming site Modified Cycle Standard
IMPORTANT Prepare separate tubes for each of the four reactions (A, C, G, and T).
Preparing 1X To prepare 1X BigDye primer reactions: Reactions

Step 1 Action Aliquot the following reagents into four PCR tubes: Reagent Ready Reaction Premix DNA template (see Template Quantity on page 4-4). Total volume 2 A (L) 4 1 C (L) 4 1 G (L) 4 1 T (L) 4 1
Preparing 2X To prepare high-sensitivity 2X BigDye primer reactions: Reactions

Step 1 Action Aliquot the following reagents into four PCR tubes: Reagent Ready Reaction Premix DNA template (see Template Quantity on page 4-4). Total Volume 2 A (L) 8 2 C (L) 8 2 G (L) 8 2 T (L) 8 2
10
10
10
10

Overview The following two protocols are used for BigDye primer cycle sequencing reactions:
o o Cycle Sequencing Using Standard Conditions Cycle Sequencing for BAC DNA
Thermal Cyclers These protocols have been optimized for all Applied Biosystems thermal cyclers:
1 2 Place the tubes in a thermal cycler and set the volume to 5 L. Repeat the following for 15 cycles: o Rapid thermal rampa to 96 C o 96 C for 10 seconds o Rapid thermal ramp to 55 C o 55 C for 5 seconds o Rapid thermal ramp to 70 C o 70 C for 1 minute 3 Repeat the following for 15 cycles: o Rapid thermal ramp to 96 C o 96 C for 10 seconds o Rapid thermal ramp to 70 C o 70 C for 1 minute 4 5 Rapid thermal ramp to 4 C and hold until ready to pool and precipitate. Continue with Preparing Extension Products for Electrophoresis on page 4-8.
Cycle Sequencing for To perform cycle sequencing for BAC DNA: BAC DNA
Step 1 2 Action Place the tubes in a thermal cycler and set the volume to 10 L. Begin thermal cycling with the following parameters: o Rapid thermal rampa to 95 C o 95 C for 5 minutes 3 Repeat the following for 20b cycles: o Rapid thermal ramp to 95 C o 95 C for 30 seconds o Rapid thermal ramp to 50 C o 50 C for 15 seconds o Rapid thermal ramp to 70 C o 70 C for 1 minute 4 Repeat the following for 15b cycles: o Rapid thermal ramp to 95 C o 95 C for 30 seconds o Rapid thermal ramp to 70 C o 70 C for 1 minute 5 6 Rapid thermal ramp to 4 C and hold until ready to pool and precipitate. Continue with Preparing Extension Products for Electrophoresis on page 4-8.
a. Rapid thermal ramp is 1 C/second. b. Increasing the number of cycles in steps 3 and 4 increases signal.

Overview Excess dye primers must be completely removed before the samples can be analyzed
by electrophoresis.
Method For BigDye primer chemistry, the standard method is ethanol precipitation without the
addition of salt. A 70% ethanol wash is optional.
IMPORTANT Use non-denatured 95% ethanol rather than absolute (100%) ethanol. Absolute ethanol absorbs water from the atmosphere, gradually decreasing its concentration. This can lead to inaccurate final concentrations of ethanol, which can affect some protocols
Ethanol You will need the following reagents and equipment for this procedure: Precipitation o Variable speed table-top centrifuge with microtiter plate tray, capable of reaching
at least 1400 g o o MicroAmp strip caps or adhesive-backed aluminum foil tape (3M Scotch Tape 431 or 439)1 95% Ethanol (ACS reagent grade), non-denatured
Note
IMPORTANT Use non-denatured 95% ethanol rather than absolute (100%) ethanol. Absolute ethanol absorbs water from the atmosphere, gradually decreasing its concentration. This can lead to inaccurate final concentrations of ethanol, which can affect some protocols.

Step 1 2 3 4 Action Remove the four 96-well plates from the thermal cycler. Remove the caps from each plate. Briefly centrifuge the plate to collect the samples at the bottom of the tubes. Using a multichannel pipet, pool the entire samples volumes from each of the four plates (i.e. containing the dideoxy A, C, G, and T reactions). Add 53 L of 95% ethanol to the pooled reactions. Seal the plate with strip caps or by applying a piece of 3M Scotch Tape 431 or 439 adhesive-backed aluminum foil tape. Press the foil onto the plate to prevent any leakage. ! WARNING CHEMICAL HAZARD. Ethanol is a flammable liquid and vapor. It may cause eye, skin, and upper respiratory tract irritation. Prolonged or repeated contact may dry skin. Exposure may cause central nervous system depression and liver damage. Please read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. 5 6 Invert the plate a few times to mix. Leave the plate on ice or at 4 C for 15 minutes to precipitate the extension products. Note Precipitation times <15 minutes will result in the loss of very short extension products. Precipitation times >24 hours will increase the precipitation of unincorporated dye terminators.
1.

Step 7 Action Place the plate in a table-top centrifuge with plate adaptor and spin it at the maximum speed, which must be 1400 g but <3000 g: o 14002000 g for 45 minutes o 20003000 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. 8 9 10 Without disturbing the precipitates, remove the adhesive tape and discard the supernatant by inverting the plate onto a paper towel. Place the inverted plate with the towel into the table-top centrifuge and spin at 700 g for 1 minute. Remove the plate and discard the paper towel. Note Pellets may or may not be visible. Vacuum drying of the samples is not necessary.
Section: Setting Up BigDye Primer Reactions for a 384-Well Format

In This Section This protocol describes how to prepare BigDye primer cycle sequencing reactions in
MicroAmp Optical 384-Well Reaction Plates.
Topic Setting Up BigDye Primer Reactions for a 384-Well Format Preparing the Reactions Performing Cycle Sequencing Preparing Extension Products for Electrophoresis See Page 4-11 4-12 4-14 4-15

This Procedure Is IMPORTANT This procedure is not recommended for 384-well plate formats. Excess BigDye Not Recommended dye primers must be completely removed by ethanol precipitation prior to electrophoresis.
However, since the capacity of the 384-well plate is 35 L, it is not possible to pool and precipitate reactions in a 384-well plate. The samples would have to be pooled and precipitated in a 96-well plate. If you wish to do this, then follow the protocol described below.
Template Quantity The table below shows recommended template quantities for the BigDye primer
chemistry.
Two Cycle The flexibility of the BigDye primer chemistry allows two options for cycle sequencing, Sequencing Options as shown in the table below.
Reaction Type 1X Template o PCR product o Plasmid o M13 0.5X o PCR product o Plasmid o M13 IMPORTANT Prepare separate tubes for each of the four reactions (A, C, G, and T). Standard Cycle Standard
Preparing 1X To prepare 1X BigDye primer reactions: Reactions

Step 1 Action Aliquot the following reagents into four PCR tubes: Reagent Ready Reaction Premix DNA template (see Template Quantity on page 4-12). Total volume 2 A (L) 4 1 C (L) 4 1 G (L) 4 1 T (L) 4 1
Preparing 0.5X To prepare 0.5X BigDye primer reactions: Reactions

Step 1 Action Dilute 5X Sequencing Buffer (400 mM Tris-HCl, 10 mM MgCl2, pH 9.0P/N 4305605, 600 reactions; 4305603, 5400 reactions) with four parts deionized water to 1X for use in this procedure. Dilute each Ready Reaction Premix (A, C, G, T) 1:1 with 1X Sequencing Buffer in a separate tube (e.g., 2 L of A Mix and 2 L of 1X Sequencing Buffer). Aliquot the following reagents into four PCR tubes for each DNA template: Reagent Diluted Ready Reaction Premix DNA template (see Template Quantity on page 4-12). Total volume 4 A (L) 4 1 C (L) 4 1 G (L) 4 1 T (L) 4 1
2 3

Overview The Cycle Sequencing Using Standard Conditions protocol is used for BigDye primer
cycle sequencing reactions.
Thermal Cycler These protocols have been optimized for the GeneAmp PCR System 9700 (in 9600
Emulation Mode).
1 2 Place the tubes in a thermal cycler and set the volume to 5 L. Repeat the following for 15 cycles: o Rapid thermal rampa to 96 C o 96 C for 10 seconds o Rapid thermal ramp to 55 C o 55 C for 5 seconds o Rapid thermal ramp to 70 C o 70 C for 1 minute 3 Repeat the following for 15 cycles: o Rapid thermal ramp to 96 C o 96 C for 10 seconds o Rapid thermal ramp to 70 C o 70 C for 1 minute 4 5 Rapid thermal ramp to 4 C and hold until ready to pool and precipitate. Continue with Preparing Extension Products for Electrophoresis on page 4-15.

Ethanol IMPORTANT This procedure is not recommended for 384-well plate formats. Because the Precipitation Not capacity of the 384-well plate is 35 L, it is not possible to pool and precipitate reactions in a Recommended 384-well plate. The samples would have to be pooled and precipitated in a 96-well plate. If you
wish to do this, refer to Preparing Extension Products for Electrophoresis on page 4-8.
dRhodamine Terminator Chemistry 5

Chapter Summary
Topic Setting Up dRhodamine Terminator Reactions for a 96-Well Format Preparing the Reactions Performing Cycle Sequencing Preparing Extension Products for Electrophoresis Setting Up dRhodamine Terminator Reactions for a 384-Well Format Preparing the Reactions Performing Cycle Sequencing Preparing Extension Products for Electrophoresis
5
See Page 5-3 5-4 5-6 5-7 5-11 5-12 5-14 5-15
dRhodamine Terminator Chemistry 5-1
5-2 dRhodamine Terminator Chemistry
Section: Setting Up dRhodamine Terminator Reactions for a 96-Well Format

In This Section This protocol describes how to prepare dRhodamine terminator cycle sequencing
Topic Setting Up dRhodamine Terminator Reactions for a 96-Well Format Preparing the Reactions Performing Cycle Sequencing Preparing Extension Products for Electrophoresis See Page 5-3 5-4 5-6 5-7

Template Quantity The table below shows recommended template quantities for dRhodamine terminator
chemistry.
Preparing Reactions To prepare dRhodamine terminator reactions:

Primer Deionized water Total Volume 2 3 Mix well and spin briefly.

Overview The following protocol is used for dRhodamine terminator cycle sequencing reactions. Thermal Cyclers These protocols have been optimized for all Applied Biosystems thermal cyclers:
Cycle Sequencing To perform cycle sequencing:

Step 1 2 Action 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. Continue with Preparing Extension Products for Electrophoresis on page 5-7.

Methods Two methods for preparing extension products for electrophoresis are presented to
offer a choice of reagents and processes.
IMPORTANT When using precipitation methods, a 70% isopropanol or 70% ethanol wash step is required. This removes residual salts and residual unincorporated dyes. If salts and unincorporated dyes are not removed from the sequencing reaction, they will compete with the extension fragments during electrokinetic injection and result in weak signals.
For dRhodamine terminator chemistries, the following methods are recommended:

Chemistry dRhodamine Terminators Recommended Methods 96-Well Reaction Plate Column Purification Ethanol/Sodium Acetate Precipitation See 5-7 5-8
96-Well Reaction For large-scale procedures, you can use the following commercially available 96-well Plate Column purification plates: Purification 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)
Refer to the manufacturers instructions procedures.
Ethanol/Sodium You will need the following reagents and equipment for this procedure: Acetate Precipitation o Variable speed table-top centrifuge with microtiter plate tray, capable of reaching
at least 1400 g o o o MicroAmp strip caps or adhesive-backed aluminum foil tape (3M Scotch Tape 431 or 439)1 3 M Sodium acetate, pH 4.6 (P/N 400320) 95% Ethanol (ACS reagent grade), non-denatured
To perform ethanol/sodium acetate precipitation:

Step 1 2 Action Remove the 96-well reaction plate 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 final ethanol concentration should be 65%. ! WARNING CHEMICAL HAZARD. Ethanol is a flammable liquid and vapor. It may cause eye, skin, and upper respiratory tract irritation. Prolonged or repeated contact may dry skin. Exposure may cause central nervous system depression and liver damage. Please read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. 3 Seal the tubes with strip caps or by applying a piece of 3M Scotch Tape 431 or 439 adhesive-backed aluminum foil tape. Press the foil onto the tubes to prevent any leakage. Invert the plate a few times to mix. Leave the plate at room temperature for 15 minutes to precipitate the extension products. Note Precipitation times <15 minutes will result in the loss of very short extension products. Precipitation times >24 hours will increase the precipitation of unincorporated dye terminators. 6 Place the plate in a table-top centrifuge with plate adaptor and spin it at the maximum speed, which must be 1400 g but <3000 g: o 14002000 g for 45 minutes o 20003000 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 plate onto a paper towel. Remove as much supernatant as possible. Rinse the pellet by adding 150 L of 70% ethanol to each well.
4 5
8
1.
To perform ethanol/sodium acetate precipitation: (continued)

Step 9 10 11 12 13 Action Seal the plates with adhesive tape and invert the plate a few times to mix. Place the plate in a table-top centrifuge and spin at 2000 g for 10 minutes. Remove the adhesive tape and discard the wash onto a paper towel that is folded to the size of the plate. Place the inverted plate with the towel into the table-top centrifuge and spin at 700 g for 1 minute. Remove the plate and discard the paper towel. Note Pellets may or may not be visible. Vacuum drying of the samples is not necessary.
Section: Setting Up dRhodamine Terminator Reactions for a 384-Well Format

In This Section This protocol describes how to prepare dRhodamine terminator cycle sequencing
Topic Setting Up dRhodamine Terminator Reactions for a 384-Well Format Preparing the Reactions Performing Cycle Sequencing Preparing Extension Products for Electrophoresis See Page 5-11 5-12 5-14 5-15

Template Quantity The table below shows recommended template quantities for dRhodamine terminator
chemistry.
Volume Capacity IMPORTANT The wells in the 384-well plates have a maximum capacity of 35 L. A portion of Restrictions this capacity will be utilized during the post-reaction clean up step. For this reason, choose your
reaction setup based on the method you will use to prepare extension products for electrophoresis. If you are purifying extension products using... 384-well plate column purification Then set up your reactions following this procedure ... o Preparing Reactions for 384-Well Plate Column Purification on page 5-13. o Preparing Reactions for Alcohol Precipitation on page 5-13. ethanol/sodium acetate precipitation Preparing Reactions for Alcohol Precipitation on page 5-13a.
a. This protocol ensures that you will not exceed the volume capacity of the 384-well plate.
Preparing Reactions Follow this reaction set up if extension products will be purified using 384-well plate for 384-Well Plate column purification. If you are purifying extension products using alcohol precipitation, Column Purification refer to Preparing Reactions for Alcohol Precipitation on page 5-13.
Primer Deionized water Total Volume 2 3 Mix well and spin briefly.
Preparing Reactions Follow this procedure if you are preparing dRhodamine terminator reactions and for Alcohol preparing the extension products for electrophoresis using any of the methods: Precipitation o 384-well plate column purification
o Ethanol/sodium acetate precipitation

Overview The following protocol is used for dRhodamine terminator cycle sequencing reactions. Thermal Cycler These protocols have been optimized for the GeneAmp PCR System 9700 (in 9600
Emulation Mode).
Cycle Sequencing To perform cycle sequencing:

Step 1 2 Action Place the tubes in a thermal cycler and set the volume to 20 L for 1X reactions or 10 L for half-volume reactions. 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. Continue with Preparing Extension Products for Electrophoresis on page 5-15.

be analyzed by electrophoresis. Excess dRhodamine terminators in sequencing reactions obscure data in the early part of the sequence and can interfere with basecalling. For an example of the effect of unincorporated dye terminators on sequencing data, refer to the Automated DNA Sequencing Chemistry Guide.
Methods Two methods for preparing extension products for electrophoresis are presented to
offer a choice of reagents and processes.
IMPORTANT When using precipitation methods, a 70% isopropanol or 70% ethanol wash step is required. This removes residual salts and residual unincorporated dyes. If salts and unincorporated dyes are not removed from the sequencing reaction, they will compete with the extension fragments during electrokinetic injection and result in weak signals.
For dRhodamine terminator chemistries, the following methods are recommended:

Chemistry dRhodamine Terminators Recommended Methods 384-Well Plate Column Purification Ethanol/Sodium Acetate Precipitation See 5-15 5-15
384-Well Plate For large-scale procedures, you can use the following commercially available 384-well Column Purification reaction plate:
o o ArrayIt (Telechem, P/N DTC-384-100) 384 System I (Edge Biosystems, P/N 95674)
Refer to the manufacturers instructions for procedures.
Ethanol/Sodium You will need the following reagents and equipment for this procedure: Acetate Precipitation o Variable speed table-top centrifuge with microtiter plate tray, capable of reaching
at least 1400 g o o o Adhesive-backed aluminum foil tape (3M Scotch Tape 431 or 439)2 3 M Sodium acetate, pH 4.6 (P/N 400320) 95% Ethanol (ACS reagent grade), non-denatured
To perform ethanol/sodium acetate precipitation:

Step 1 Action Remove the 384-well reaction plate from the thermal cycler. Remove the seal from each tube.
2.
To perform ethanol/sodium acetate precipitation: (continued)

Step 2 Action Add the following: o 1.0 L of 3 M sodium acetate (NaOAc), pH 4.6 o 18 L of 95% ethanol (EtOH) The final ethanol concentration should be 60 3%. ! WARNING CHEMICAL HAZARD. Ethanol is a flammable liquid and vapor. It may cause eye, skin, and upper respiratory tract irritation. Prolonged or repeated contact may dry skin. Exposure may cause central nervous system depression and liver damage. Please read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. 3 4 5 Seal the tubes by applying a piece of 3M Scotch Tape 431 or 439 adhesive-backed aluminum foil tape. Press the foil onto the tubes to prevent any leakage. Invert the plate a few times to mix. Leave the plate at room temperature for 15 minutes to precipitate the extension products. Note Precipitation times <15 minutes will result in the loss of very short extension products. Precipitation times >24 hours will increase the precipitation of unincorporated dye terminators. 6 Place the plate in a table-top centrifuge with plate adaptor and spin it at the maximum speed, which must be 1400 g but <3000 g: o 14002000 g for 45 minutes o 20003000 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 plate onto a paper towel. Remove as much supernatant as possible. Rinse the pellet by adding 35 L of 70% ethanol to each well. Seal the plates with adhesive tape and invert the plate a few times to mix. Place the plate in a table-top centrifuge and spin at 2000 g for 10 minutes. Remove the adhesive tape and discard the wash onto a paper towel that is folded to the size of the plate. Place the inverted plate with the towel into the table-top centrifuge and spin at 700 g for 1 minute. Remove the plate 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
Sample Injections
Chapter Summary
Topic Preparing Samples for Injection Centrifuging Samples Optimizing Electrokinetic Injection Optimizing Electrophoresis Conditions
6
See Page 6-2 6-3 6-4 6-6
Sample Injections 6-1
Preparing Samples for Injection

Injection Solutions Before DNA samples and standards can be placed on the ABI PRISM 3100 Genetic
Analyzer, they must be resuspended in formamide.
IMPORTANT Covered samples can be stored in formamide for 24 hours at room temperature. Uncovered samples can only be stored in formamide for 8 hours. ! WARNING CHEMICAL HAZARD. Formamide is harmful if absorbed through the skin and may cause irritation to the eyes, skin, and respiratory tract. It may cause damage to the central nervous system and the male and female reproductive systems, and is a possible birth defect hazard. Please read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.
Sample Volumes for IMPORTANT Always stay within the volume range specified below. Reaction Plates
For correct delivery of samples to the capillary tips, the sample volumes in the 96-well reaction plate wells must remain within the ranges specified in the table below. For 96-Well Plates
Type of Reaction Plate 96-well 96-well Minimum Volume (L) 10.0 10.0 Maximum Volume (L)a 150 150 Recommended Volume (L) 1030 1030
Reaction Type 1X High-Sensitivity (2X)
a. The maximum volume is to ensure that the septa does not touch the sample and cause cross-contamination.
For 384-Well Plates

Type of Reaction Plate 384-well Minimum Volume (L) 10.0 Maximum Volume (L)a 15 Recommended Volume (L) 1015
Reaction Type For 384-well column purification For precipitation
384-well
10.0
15
1015
a. The maximum volume is to ensure that the septa does not touch the sample and cause cross-contamination.
Covering Sample Samples in injection solution are subject to evaporation or degradation depending on Plates the injection solution used. The longer the samples are exposed to air, the more likely
this problem will occur. To avoid issues with evaporation or degradation, Applied Biosystems recommends immediately covering the sample plates with the provided septa.
6-2 Sample Injections
Centrifuging Samples
For 96 and 384-Well Before placing your plates on the autosampler, you must centrifuge them to bring the Plates samples down to the bottom of the tubes or wells. Failure to do this properly will result
in the samples not being injected into the capillaries. To centrifuge your plates:
Step 1 2 3 Action Seal the plates. Centrifuge the 96- or 384-well reaction plates at 2000 x g for 1 minute. Hold the plates up to the light and examine them carefully to make sure that every sample is positioned at the bottom of the tube or well. Before you place your plates on the plate deck, the samples in your tubes or wells should: Look like this... Not look like this... not look like this...
The sample is positioned correctly in the bottom of the tube or well.
The sample lies on the side wall because the plate or tube was not centrifuged.
An air bubble lies at the bottom of the tube or well because the sample was not: o Centrifuged with enough force, or o Centrifuged for enough time
If one or more samples are not positioned correctly, repeat step 2. 4 Your samples are now ready to be placed on the plate deck.
Optimizing Electrokinetic Injection

Overview Optimizing electrokinetic injection can greatly improve data quality and run-to-run
reproducibility. The goal is to inject sufficient DNA to yield peaks of adequate height (i.e., data with a good signal-to-noise ratio) while maintaining resolution and read length. The 3100 Genetic Analyzer run modules have preset values for injection times and voltages. These values are adequate for most applications. However, you should consider modifying the injection parameters when: o o o The signal is too strong The signal is too weak The resolution is poor.
IMPORTANT For information on setting electrokinetic injection values, refer to the ABI PRISM 3100 Genetic Analyzer Users Manual.
Signal Too Strong If the signal is too strong:

o o o Decrease the injection time. Decrease the injection voltage. Decrease the concentration of DNA fragments in the sample.
Signal Too Weak If the signal is too weak when using the 50-cm array: Using 50-cm Array o Increase the injection time. See the table below.
Injection Time (seconds) 20 40 80 Total EKIa Product (V-sec/cm) 500 1000 2000
Volts/cm 25 25 25
Description Default setting for injection time Maximum injection time recommended with minimal effect on resolution Signal strength increases at this injection time, but at the expense of resolution
a. electrokinetic injection (EKI)
o o o
Reduce the amount of salt in the sample. Increase the concentration of the DNA extension products. Do not increase the voltage. Increasing the voltage increases the signal, but may reduce resolution across the array. It is better to adjust the injection time in order to increase signal.
IMPORTANT Negative ions, e.g., EDTA and acetate, compete with DNA for injection. To reduce the amount of salt in a sequencing reaction, use column purification.
Signal Too Weak If the signal is too weak when using the 36-cm array: Using 36-cm Array o Increase the injection time. See the table below.
Injection Time (seconds) 15 25 50 Total EKIa Product (V-sec/cm) 480 800 1600
Volts/cm 32 32 32
Description Default setting for injection time Maximum injection time recommended with minimal effect on resolution Signal strength increases at this injection time, but at the expense of resolution
a. electrokinetic injection (EKI)
o o o
Reduce the amount of salt in the sample. Increase the concentration of the DNA extension products. Do not increase the voltage. Increasing the voltage increases the signal, but may reduce resolution across the array. It is better to adjust the injection time in order to increase signal.
IMPORTANT Negative ions, e.g., EDTA and acetate, compete with DNA for injection. To reduce the amount of salt in a sequencing reaction, use column purification.
Poor Resolution If the resolution is poor:

o Decrease the injection time. Decreasing the injection time will decrease the signal strength. To compensate for the loss in signal, lower the salt concentration in the sample or increase the DNA extension product concentration. o Decrease the run voltage by approximately 15%.
Optimizing Electrophoresis Conditions

Overview Optimizing electrophoresis conditions (run time, run voltage, and run temperature)
can greatly improve data quality, run-to-run reproducibility, and/or throughput. When selecting values for these parameters, consider the following factors: o o Read length desired Required degree of resolution
Run Time Determining Required Run Time

To ensure that you collect sufficient data to perform analysis, set the electrophoresis run time approximately 510 minutes higher than the migration time of the longest fragment you want to detect. To change the data collection time refer to:
Topic Changing Run Time for the 50-cm Array Changing Run Time for the 36-cm Array See Page 6-6 6-7
Changing Run Time for the 50-cm Array You can change the data collection time for special requirements. For example, you can shorten the data collection time if you only need information about short extension products (e.g., in PCR sequencing). Figure 6-1 illustrates the amount of time that elapses before a DNA fragment traveling through a 50-cm capillary reaches the fluorescence detector and data collection begins. The graph assumes the following information: o o o o The run module is programmed for 41 minutes of protocol events, including 20 minutes of programmed delay time, before data collection starts. The separation voltage is 12,200volts. The separation distance is 50 cm with a total capillary length of 61 cm. The temperature is 50 C.
Collection Time (Minutes)
Fragment Size Collected (Basepairs) Figure 6-1 Graph of the time required for fragments to reach the detector using a 50-cm array
Changing Run Time for the 36-cm Array You can change the data collection time for special requirements. For example, you can shorten the data collection time if you only need information about short extension products (e.g., in PCR sequencing). Figure 6-2 illustrates the amount of time that elapses before a DNA fragment traveling through a 36-cm capillary reaches the fluorescence detector and data collection begins. The graph assumes the following information: o o o o The run module is programmed for 26 minutes of protocol events, including 4 minutes of programmed delay time, before data collection starts. The separation voltage is 15,000 volts. The separation distance is 36 cm with a total capillary length of 47 cm. The temperature is 55 C.
Collection Time (Minutes)
Fragment Size Collected (Basepairs) Figure 6-2 Graph of the time required for fragments to reach the detector using 36-cm array.
Run Temperature Run Temperature and Run Voltage Protocols for sequencing applications with 3100 POP-6 specify a 5055 C
electrophoresis temperature. For templates that do not denature readily at 50 C, the run temperature can be increased up to 60 C. Run Voltage Decreased run voltage or temperature decreases the migration rate of fragments. Longer run times are required to collect the same size fragments as in standard conditions. The basecaller may not be able to analyze the spacing values. Increased run voltage or temperature increases migration rates, allowing for shorter run times, but decreased resolution.
Laboratory The laboratory temperature should be maintained between 15 and 30 C. It should not Temperature and fluctuate more than 6 C during a run for optimal results. Humidity
The ABI PRISM 3100 Genetic Analyzer can tolerate up to 80% non-condensing relative humidity. Avoid placing the instrument near heaters, cooling ducts, windows, or back-to-back with another instrument.
For More For information on setting electrophoresis parameters, refer to the ABI PRISM 3100 Information Genetic Analyzer Users Manual.
Purchasing or Preparing Formamide A

Deionizing and Storing Formamide
Formamide, Formamide is used to denature the DNA samples before placing them on the Denaturation Agent ABI PRISM 3100 Genetic Analyzer. Option to Purchase There are two ways to obtain formamide for use with the 3100 Genetic Analyzer. You or to Make can:
o o Prepare it yourself, using a mixed-bed (anionic and cationic) ion-exchange resin, as described in the directions below. Purchase Hi-Di Formamide from Applied Biosystems.
Purchasing Hi-Di Hi-Di Formamide is suitable for use with the 3100 Genetic Analyzer. It is available Formamide from from Applied Biosystems in 25-mL bottles (P/N 4311320). Applied Biosystems The Problem with Formamide purchased from commercial suppliers is often contaminated with variable Commercial amounts of water and undesirable organic and inorganic ions. In addition, formamide Formamide is often supplied in glass bottles, which, when opened, exposes the formamide to the
air and allow it to absorb water. Water reacts slowly with formamide to produce formic acid (methanoic acid) and ammonia. The ionic products of this reaction cause two problems: o o They compete significantly with the larger DNA ions for injection into the capillary, resulting in weaker signals. They react with the DNA, causing degradation of the sample.
Figure A-1shows the effect of formamide exposure to the air on electropherogram data.
Purchasing or Preparing Formamide A-1
Figure A-1 The effect of exposing formamide-resuspended samples to air. The top panel shows electropherogram data from samples incubated for 51 hours with a lid. The bottom panel shows electropherogram data from samples incubated for 48 hours with no lid.
A-2 Purchasing or Preparing Formamide
Materials Required The following materials are recommended for this procedure:
Material Formamide Description The raw (prior to deionization) formamide should be: o 99.5% purity or greater, with low water content o Packed under an inert gas o Have a conductivity of approximately 100 Siemens/cm or less Note Siemens, formerly called mho, are the units of measurement for specific conductance or conductivity. Ion-exchange resin o Mixed-bed resin containing the following strong ion exchange functional groups: R-SO3- (as H form) (cation) R-CH2N+(CH3)3, (as OH form) (anion) These groups are attached to a styrene divinylbenzene matrix with 8% cross-linkage. o The minimum wet capacity is 1.5 meq/mL with 2050 dry mesh size (AG501 X8, molecular biology grade mixed-bed resin) o Available from Bio-Rad Laboratories (P/N 143-6424) or equivalent Conductivity meter A commercial conductivity meter, or pH meter with an external conductivity cell, is sufficient to measure the conductivity of formamide if it has a cell constant of 1.0 o Dihydrate (Mr 372.2) o ACS reagent, 99% purity or greater o Available from Sigma (P/N E4884) or equivalent Container for storing formamide Use a polypropylene screw-cap container Note Glass containers are not recommended because of potential contamination from minerals.
+
Na2EDTA
Ion-Exchange Resin The raw formamide is deionized with cationic and anionic mixed resins to remove
impurities such as ammonium and formate ions. Deionization occurs at a slow masstransfer rate in the equilibrium ion exchange kinetics due to: o o Physical changes in the resin in the presence of formamide Differences in molecular size and selectivity between the impurity ions and the + H and OH counterions.
Therefore, the conductivity of formamide must be monitored over time to determine the extent of deionization by the resin.
Calibrating the A conductivity meter and cell are needed to measure the effectiveness of the Conductivity Meter deionization process. The more deionized the formamide, the lower its conductivity.
Within the range or measurement, the conductivity meter should be routinely calibrated (to 50 Siemens/cm or less). Calibrate the meter using standard solutions that are traceable to the National Institute of Standards and Technology (NIST). Because temperature affects conductivity, samples must be brought to room temperature before measuring the conductivity.
Preparing EDTA Alkaline EDTA (ethylenediaminetetraacetic acid) is added to the deionized formamide
to stabilize it and to facilitate the electrokinetic injection of DNA. To minimize the amount of water added to the formamide, a concentrated (200-mM) stock solution of the EDTA is added. To prepare the 200-mM EDTA stock solution:
Step 1 2 Action Add 7.44 g of Na2EDTA to 70 mL of deionized water and stir. While stirring, slowly adjust to pH 8.08.8 by dropwise addition of a concentrated solution of sodium hydroxide. ! WARNING CHEMICAL HAZARD. Sodium hydroxide (NaOH) causes severe burns to the skin, eyes, and respiratory tract. Please read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. Note This helps the EDTA to dissolve over time, because the EDTA has a limited solubility until the pH is increased. 3 4 Dilute to 100 mL with deionized water. Store at 4 C.
Procedure
! WARNING CHEMICAL HAZARD. Formamide is harmful if absorbed through the skin and may cause irritation to the eyes, skin, and respiratory tract. It may cause damage to the central nervous system and the male and female reproductive system, and is a possible birth defect hazard. Please read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. Note There is not a stopping point in this procedure. Complete the procedure from resin washing to freezing the formamide, without interruption.
To begin purification and measure conductivity:

Step 1 2 Action Calibrate the conductivity meter cell, and rinse the cell with distilled water. In a polypropylene screw-cap container, wash 10 g of Bio-Rad AG501 X8 ion-exchange resin by swirling the sample with 1020 mL of formamide for 1 minute. Either decant off or filter through a course nylon or teflon filter, and discard the formamide. Repeat steps 2 and 3 twice. Add 100 mL of formamide to the washed resin. Cap the mixture, ensuring that it is well sealed.
3 4 5 6
A-4 Purchasing or Preparing Formamide
To begin purification and measure conductivity: (continued)

Step 7 Action Stir the mixture rapidly with a magnetic stirrer, or mix with an electric shaker, ensuring that the resin is suspended and mixes thoroughly with the formamide. Stir at room temperature for approximately 2 hours. Stop stirring and allow the resin to settle for 5 minutes. Remove a small aliquot of the mixture, and measure the conductivity at room temperature. Rinse the conductivity cell with distilled water. If the conductivity is... >5 Siemens/cm <5 Siemens/cm Then... Return to step 7, stirring for an additional 30 minutes. Continue with To complete purification of deionized formamide:..
8 9 10 11
Note If the conductivity is not <5 Siemens/cm after about 4.5 hours of mixing, repeat the entire procedure using a new lot of formamide and new resin. Note Starting formamide with a higher purity and lower conductivity deionizes more efficiently.
To complete purification of deionized formamide:

Step 1 2 3 Action Vacuum-filter the deionized formamide using a 0.2- or 0.4-m nylon or teflon filter. Measure the final volume of deionized formamide. Add the required volume of 200-mM EDTA to the deionized formamide to achieve a final concentration of approximately 0.3-mM EDTA. Note After adding the EDTA, the final conductivity of the formulation is increased to approximately 25 Siemens/cm. Use the equation below to calculate the volume of EDTA to add. VEDTA (L) = 1.5V Form(mL) Where, VEDTA(L) = volume of EDTA to add in microliters VFORM(mL)= measured volume of formamide in milliliters Sample calculation with a final volume of 90-mL formamide: VEDTA(L) = 1.5 90 = 135 L 4 Immediately aliquot the formamide into smaller polypropylene tubes and store at 15 to 20 C for up to about 6 months.
Using the When ready for use, thaw and completely use one tube at a time before opening and Formamide exposing another. Store the tubes at 4 C during the day for intermittent use.
Otherwise, refreeze them. Minimize the number of freeze-thaw cycles for each tube.
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Troubleshooting
Chemistry Troubleshooting
Troubleshooting Some common observations associated with the chemistries used on the ABI PRISM Table 3100 Genetic Analyzer are listed below.
Observation Poor data resolution Possible Cause Clogged capillary caused by an excess of protein, template, or other sample impurities Samples stored in formamide for longer than 24 hours at room temperature. Overloading of the sample Excess DNA injected into the capillary Recommended Action Replace the array.
Re-prepare the samples.
Dilute the sample Adjust the injection parameter. Refer to Optimizing Electrokinetic Injection on page 6-4. Refer to this chemistry guide for a description of recommended template quantities. If possible, resuspend the template in a smaller volume. Increase injection time. Refer to Optimizing Electrokinetic Injection on page 6-4.
Weak signal
The quantity of template or primers in the sequencing reaction or the quantity of sample injected is too low.
Excess salt is present in the sample. Samples stored in formamide for longer than 24 hours at room temperature.
Clean up the sample using a spin column or a 70% ethanol wash. Re-prepare the samples.
Troubleshooting C-1
Observation High background
Possible Cause Dirty template
Recommended Action Refer to the Automated DNA Sequencing Chemistry Guide for a description of how to clean up dirty templates. Refer to this chemistry guide for a description of recommended template quantities.
Top-heavy samples
Amount of template in the sequencing reaction is too high, creating an excess of short fragments that are preferentially injected into the capillary. The concentration of extension products is too high.
Dilute the sample or decrease the injection time. Clean up the DNA sample and repeat the cycle sequencing reaction.
Blank lanes or no signal
Blocked capillary caused by an excess of protein, template, or other impurities, or by dried polymer. Cycle sequencing reaction failed.
Breakdown of BigDye G nucleotide. Refer to The Problem with Commercial Formamide on page A-1.
Formamide degradation caused by exposure to the air.
Use formamide as recommended in Appendix A, Purchasing or Preparing Formamide. Cover plates with septa. Try using an alternative injection solution.
C-2 Troubleshooting
Index
Numerics
3100 Genetic Analyzer capillary electrophoresis, optimizing 6-6 to 6-9 chemistries supported 2-2 overview 2-2 to 2-4 performance, factors affecting 2-6 plates, compatible 2-10 run cycle overview 2-3 3100 POP-6 polymer, See POP-6 polymer 36-cm array changing run time 6-7 optimizing electrophoresis 6-6 384-well format BigDye primer reactions 4-11 to 4-15 BigDye terminator reactions 3-13 to 3-21 dRhodamine terminator reactions 5-11 to 5-16 384-well plate centrifuging samples 6-3 50-cm array changing run time 6-6 optimizing electrokinetic injection 6-4 optimizing electrophoresis 6-6 96-well format BigDye primer reactions 4-3 to 4-9 BigDye terminator reactions 3-3 to 3-12 dRhodamine terminator reactions 5-3 to 5-9 96-well plate centrifuging samples 6-3 recommended volume 6-2 ethanol precipitation 4-8 extension products, preparing for electrophoresis 4-8 reactions, preparing 4-4 to 4-5 template quantity 4-4 BigDye terminator reactions 384-well format 3-13 to 3-21 1X reactions 3-15 control DNA 3-14 cycle sequencing 3-17 cycle sequencing options 3-15 ethanol precipitation 3-19 extension products, preparing for electrophoresis 3-18 to 3-21 isopropanol precipitation 3-20 modifications to thermal cycling program 3-17 preparing reactions for precipitation 3-16, 5-13 reactions, preparing 3-14 to 3-16 template quantity 3-14 volume restrictions 3-14 96-well format 3-3 to 3-12 1X reactions 3-5 2X reactions for BACs, YACs, Cosmids 3-6 2X reactions for genomic DNA 3-6 control DNA 3-4 cycle sequencing 3-6 cycle sequencing options 3-5 ethanol precipitation 3-11 extension products, preparing for electrophoresis 3-9 to 3-12 isopropanol precipitation 3-10 modifications to thermal cycling program 3-7 reactions, preparing 3-4 to 3-6 template quantity 3-4
B
Bacterial Artificial Chromosome (BAC), cycle sequencing 3-8 bacterial genomic DNA, cycle sequencing 3-8 bases, compatible 2-10 BigDye primer reactions 384-well format 4-11 to 4-15 0.5X reactions 4-13 1X reactions 4-13 control DNA 4-12 cycle sequencing 4-14 ethanol precipitation 4-15 extension products, preparing for electrophoresis 4-15 reaction dilutions 4-12 reactions, preparing 4-12 to 4-13 template quantity 4-12 volume restrictions 4-12, 4-15 96-well format 4-3 to 4-9 1X reactions 4-5 2X reactions 4-5 control DNA 4-4 cycle sequencing for BAC DNA 4-7 cycle sequencing, standard conditions 4-6
C
capillary electrophoresis, See electrophoresis centrifuging 384-well plates 6-3 96-well plates 6-3 chemistry BigDye primer, 384-well format 4-11 to 4-15 cycle sequencing 4-14 preparing extension products 4-15 preparing reactions 4-12 to 4-13 sample clean-up 4-15 BigDye primer, 96-well format 4-3 to 4-9 cycle sequencing 4-6 to 4-7 preparing extension products 4-8 to 4-9 preparing reactions 4-4 to 4-5 reaction clean-up 4-8 to 4-9 BigDye terminator, 384-well format 3-13 to 3-21 cycle sequencing 3-17 extension products, preparing for
Index-1
electrophoresis 3-18 to 3-21 preparing reactions 3-14 to 3-16 reaction clean-up 3-18 to 3-21 BigDye terminator, 96-well format 3-3 to 3-12 extension products, preparing for electrophoresis 3-9 to 3-12 cycle sequencing 3-7 preparing reactions 3-4 to 3-6 reaction clean-up 3-9 to 3-12 dRhodamine terminator, 384-well format 5-11 to 5-16 cycle sequencing 5-14 extension products, preparing for electrophoresis 5-15 to 5-16 preparing reactions 5-12 to 5-13 reaction clean-up 5-15 to 5-16 dRhodamine terminator, 96-well format 5-3 to 5-9 cycle sequencing 5-6 extension products, preparing for electrophoresis 5-7 to 5-9 preparing reactions 5-4 to 5-5 reaction clean-up 5-7 to 5-9 troubleshooting C-1 to C-2 types supported 2-2 conductivity meter, for deionizing formamide A-4 customer support e-mail address B-1 help B-1 to B-5 Internet address B-5 telephone/fax B-2 to B-4 cycle sequencing 384-well format BigDye primer reactions 4-14 BigDye terminator reactions 3-17 dRhodamine terminator reactions 5-14 96-well format BigDye primer reactions 4-6 BigDye terminator reactions 3-7 dRhodamine terminator reactions 5-6
preparing reactions 5-4 to 5-5 template quantity 5-4 dye terminator chemistries See BigDye terminator reactions 5-3 See dRhodamine terminator reactions 5-3
E
electrophoresis advantages of capillary 2-5 capillary versus slab gel 2-4 optimizing 6-6 to 6-9 e-mail, address for technical support ethanol precipitation BigDye primer reactions 384-well format 4-15 96-well format 4-8 BigDye terminators 384-well format 3-19 96-well format 3-11 ethanol/sodium acetate precipitation 384-well format 5-15 96-well format 5-8 extension products, preparing BigDye primer 384-well 4-15 96-well 4-8 to 4-9 BigDye terminator 384-well 3-18 to 3-21 96-well 3-9 to 3-12 dRhodamine terminator 384-well 5-15 to 5-16 96-well 5-7 to 5-9
B-1
F
formamide definition A-1 deionizing A-1 to A-5 effects of air exposure A-1 to A-2 problem with commercial types A-1 purchasing Hi-Di formamide A-1 storing A-5 storing samples in 6-2
D
deionized formamide See formamide detergent, effect on template quality 2-7 Documents on Demand B-5 dRhodamine terminator reactions 384-well format 5-11 to 5-16 ethanol/sodium acetate precipitation extension products, preparing for electrophoresis 5-15 reactions, preparing 5-12 to 5-13 template quantitiy 5-12 volume capacity restrictions 5-12 96-well format 5-3 to 5-9 control DNA 5-4 cycle sequencing 5-5 ethanol/sodium acetate precipitation extension products, preparing for electrophoresis 5-7 to 5-9
G
gel electrophoresis, See electrophoresis 5-15
H
help e-mail address B-1 Internet address B-5 telephone hours B-1 telephone/fax B-2 to B-4 humidity, in the laboratory 6-9 5-8
I
injecting samples
Index-2
384-well format 6-3 96-well format 6-2 injection time 6-4 injection voltage 6-4 instrument chemistries supported 2-2 factors affecting performance 2-6 overview 2-2 to 2-4 plates, compatible 2-10 run cycle 2-3 Internet address Documents on Demand B-5 ion exchange resin, for deionizing formamide A-3 isopropanol precipitation 384-well format 3-20 96-well format 3-10
L
laboratory humidity 6-9 laboratory temperature 6-9
1X reaction 4-5 2X reaction 4-5 BigDye terminator, 384-well for plate column purification 3-15 for precipitation 3-16 BigDye terminator, 96-well 1X reaction 3-5 2X for BACs, PACs, YACs 3-6 2X for bacterial genomic DNA 3-6 dRhodamine terminator, 96-well 1X reaction 5-5 resolution, poor, how to resolve 6-5 rhodamine dye terminators, See dRhodamine terminator reactions RNA, effect on template quality 2-7 run temperature 6-8 run time changing for 36-cm array 6-7 changing for 50-cm array 6-6 run voltage 6-8
M
manuals, part numbers 1-2 MicroAmp 384-Well Reaction Plate 2-10 MicroAmp Optical 96-Well Reaction Plate 2-10 modifications, to thermal cycling protocol 3-7
S
safety 1-3 See also warnings salt, effect on template quality 2-6 sample injections 384-well format 6-3 96-well format 6-2 signal, too strong 6-4 signal, too weak 6-4 spin column, See plate column purification
P
PAC, cycle sequencing 3-8 part numbers cycle sequencing kits 2-2 formamide deionization reagents A-3 Hi-Di formamide A-1 POP-6 polymer 2-5 users manuals 1-2 plate column purification 384-well 3-18 96-well 3-9 plates 384-well 2-10 96-well 2-10 polymer See POP-6 polymer POP-6 polymer about 2-4 part number 2-5 run temperature 6-8 run voltage 6-8 primers, modifying cycling parameters based on protein, effect on template quality 2-7
T
technical support B-1 to B-5 e-mail address B-1 Internet address B-5 telephone/fax B-2 to B-4 temperature in the laboratory 6-9 of the run 6-8 template quality residual detergent effect 2-7 residual protein effect 2-7 residual RNA effect 2-7 residual salts effect 2-6 template quantity BigDye primer reactions 384-well format 4-12 96-well format 4-4 BigDye terminator reactions 384-well format 3-14 96-well format 3-4 dRhodamine terminator reactions 384-well format 5-12 96-well format 5-4 effect of excess 2-8 template, modifying cycling parameters based on terminator reactions
3-7
R
reactions, preparing BigDye primer, 384-well 0.5X reaction 4-13 1X reaction 4-13 BigDye primer, 96-well
3-7
Index-3
See BigDye terminator reactions See dRhodamine terminator reactions thermal cyclers for 384-well format 3-17 for 96-well format 3-7 thermal cycling, modifications to BigDye terminator 3-7 troubleshooting C-1 to C-2
U
users manuals, part numbers 1-2
V
voltage, injection 6-4 voltage, run 6-8 volume restrictions, 384-well format BigDye primer reactions 4-12, 4-15 BigDye terminator reactions 3-14 dRhodamine terminator reactions 5-12 volumes, sample for 384-well plate 6-2 for 96-well plate 6-2
W
WWW address Applied Biosystems B-5 Documents on Demand B-5
Y
YAC, cycle sequencing 3-8
Index-4
Headquarters 850 Lincoln Centre Drive Foster City, CA 94404 USA Phone: +1 650.638.5800 Toll Free: +1 800.345.5224 Fax: +1 650.638.5884 Worldwide Sales Offices Applied Biosystems vast distribution and service network, composed of highly trained support and applications personnel, reaches into 150 countries on six continents. For international office locations, please call our local office or refer to our web site at www.appliedbiosystems.com.
www.appliedbiosystems.com
Applera Corporation is committed to providing the worlds leading technology and information for life scientists. Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses. Printed in the USA, 01/2001 Part Number 4315831B
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ABI PRISM® 3100 Genetic Analyzer-Sequencing

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ABI PRISM 3100 Genetic Analyzer

Sequencing Chemistry Guide

2 About the 3100 Genetic Analyzer

3 BigDye Terminator Chemistry

Section: Setting Up BigDye Terminator Reactions for a 96-Well Format . . . . . . . . .3-3

Section: Setting Up BigDye Terminator Reactions for a 384-Well Format . . . . . . .3-13

Ethanol Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-19 Isopropanol Precipitation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-20

4 BigDye Primer Chemistry

Section: Setting Up BigDye Primer Reactions for a 96-Well Format . . . . . . . . . . . . 4-3

Section: Setting Up BigDye Primer Reactions for a 384-Well Format . . . . . . . . . . 4-11

5 dRhodamine Terminator Chemistry

Section: Setting Up dRhodamine Terminator Reactions for a 96-Well Format . . . .5-3

Section: Setting Up dRhodamine Terminator Reactions for a 384-Well Format . .5-11

Manual Overview 1-1

About This Manual

1-2 Manual Overview

Manual Overview 1-3

1-4 Manual Overview

Use Documents on Demand on B-5. Dial 1-800-327-3002, then press 1.

To order in... English French

By telephone from any other country

See Regional Offices Sales and Service on B-4.

Manual Overview 1-5

About the 3100 Genetic Analyzer 2

About the 3100 Genetic Analyzer 2-1

2-2 About the 3100 Genetic Analyzer

Run Cycle Overview

About the 3100 Genetic Analyzer 2-3

2-4 About the 3100 Genetic Analyzer

About the 3100 Genetic Analyzer 2-5

Factors Affecting Instrument Performance

2-6 About the 3100 Genetic Analyzer

The effect of salt during electrokinetic injection

About the 3100 Genetic Analyzer 2-7

Template Quantity Excess Template in Your Sample

2-8 About the 3100 Genetic Analyzer

Electropherogram from the sample run in Figure 2-2

About the 3100 Genetic Analyzer 2-9

MicroAmp Optical 96-Well Reaction Plate

MicroAmp 384-Well Reaction Plate

2-10 About the 3100 Genetic Analyzer

BigDye Terminator Chemistry

BigDye Terminator Chemistry 3-1

3-2 BigDye Terminator Chemistry

Section: Setting Up BigDye Terminator Reactions for a 96-Well Format

BigDye Terminator Chemistry 3-3

Preparing the Reactions

3-4 BigDye Terminator Chemistry

Preparing 1X To prepare 1X BigDye terminator reactions: Reactions

Continue with Performing Cycle Sequencing on page 3-7.

BigDye Terminator Chemistry 3-5

3-6 BigDye Terminator Chemistry

Performing Cycle Sequencing

a. Rapid thermal ramp is 1 C/second.

BigDye Terminator Chemistry 3-7

3-8 BigDye Terminator Chemistry

Preparing Extension Products for Electrophoresis

For BigDye terminator chemistries, the following methods are recommended:

Automated DNA Sequencing Chemistry Guide

96-Well Plate Column Purification Isopropanol Precipitation Ethanol Precipitation

Refer to the manufacturers instructions procedures.

BigDye Terminator Chemistry 3-9

To perform isopropanol precipitation: