Automated DNA Sequencing Chemistry Guide © Copyright 2000, Applied Biosystems For Research Use Only. Not for use in diagnostic procedures. ABI PRISM and its design, Applied Biosystems, and MicroAmp are registered trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries. ABI, BigDye, CATALYST, POP, POP-4, POP-6, and Primer Express are 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. Long Ranger is a trademark of The FMC Corporation. Macintosh and Power Macintosh are registered trademarks of Apple Computer, Inc. pGEM is a registered trademark of Promega Corporation. Contents 1 Introduction. 1-1 New DNA Sequencing Chemistry Guide . 1-1 Introduction to Automated DNA Sequencing . 1-2 ABI PRISM Sequencing Chemistries . 1-5 Applied Biosystems DNA Sequencing Instruments. 1-7 Data Collection and Analysis Settings . 1-12 2 ABI PRISM DNA Sequencing Chemistries . 2-1 Overview . 2-1 Dye Terminator Cycle Sequencing Kits . 2-2 Dye Primer Cycle Sequencing Kits . 2-8 Dye Spectra . 2-12 Chemistry/Instrument/Filter Set Compatibilities . 2-13 Dye/Base Relationships for Sequencing Chemistries . 2-14 Choosing a Sequencing Chemistry. 2-15 3 Performing DNA Sequencing Reactions . 3-1 Overview . 3-1 DNA Template Preparation . 3-2 Sequencing PCR Templates . 3-10 DNA Template Quality. 3-15 DNA Template Quantity. 3-17 Primer Design and Quantitation . 3-18 Reagent and Equipment Considerations. 3-20 Preparing Cycle Sequencing Reactions . 3-21 Cycle Sequencing . 3-27 Preparing Extension Products for Electrophoresis. 3-33 Removing Unincorporated Dye Terminators . 3-34 Preparing Dye Primer Reaction Products for Electrophoresis . 3-46 Preparing and Loading Samples for Gel Electrophoresis. 3-50 Preparing and Loading Samples for Capillary Electrophoresis . 3-53 4 Optimizing Gel Electrophoresis . 4-1 Introduction . 4-1 Reagents . 4-2 i Avoiding Problems with Sequencing Gels . 4-4 5 Optimizing Capillary Electrophoresis . 5-1 Introduction . 5-1 Capillary Electrophoresis Consumables . 5-2 Optimizing Electrokinetic Injection . 5-4 Optimizing Electrophoresis Conditions. 5-7 Run Parameters for Specific Sequencing Chemistries. 5-8 6 Optimizing Software Settings. 6-1 Introduction . 6-1 Choosing a Run Module . 6-2 Choosing a Dye Set/Primer (Mobility) File. 6-3 Choosing the Correct Basecaller . 6-6 Creating an Instrument (Matrix) File. 6-7 Setting the Data Analysis Range . 6-15 7 Data Evaluation and Troubleshooting . 7-1 Overview . 7-1 Data Evaluation . 7-2 Practical Examples of Data Evaluation . 7-10 Troubleshooting Sequencing Reactions. 7-16 Troubleshooting DNA Sequence Composition Problems . 7-30 Troubleshooting Sequencing Data. 7-39 Troubleshooting Gel Electrophoresis on the ABI 373 and ABI PRISM 377 . 7-44 Troubleshooting Capillary Electrophoresis on the ABI PRISM 310. 7-55 Troubleshooting Software Settings . 7-62 A Gel Preparation . A-1 Introduction . A-1 Protocol and Run Conditions for 19:1 Polyacrylamide Gels . A-2 Protocol and Run Conditions for 29:1 Polyacrylamide Gels. A-6 Protocol and Run Conditions for Long Ranger and PAGE-PLUS Gels . A-10 Preparing APS, TBE Buffer, and Deionized Formamide . A-15 ii B IUB Codes . .B-1 C References . .C-1 D Technical Support . .D-1 To Reach Us on the Web. D-1 Hours for Telephone Technical Support . D-1 To Reach Us by Telephone or Fax in North America. D-1 Documents on Demand . D-2 To Reach Us by E-Mail . D-3 Regional Offices Sales and Service . D-3 E Part Numbers. .E-1 ABI PRISM DNA Sequencing Kits and Reagents. .E-1 ABI PRISM 310 Genetic Analyzer . ..