ABI P RISM - Applied Biosystems

1 ABI PRISM . 310 Genetic Analyzer User's Manual Copyright 2001, 2010 Applied Biosystems For Research Use Only. Not for use in diagnostic procedures. NOTICE TO PURCHASER: This instrument, Serial No. _____, is Authorized for use in DNA sequencing and fragment analysis. This authorization is included in the purchase price of this instrument and corresponds to the up-front fee component of a license under process claims of patents and under all process claims for DNA sequence and fragment analysis of patents now or hereafter owned or licensable by Applied Biosystems for which an Authorization is required, and under corresponding process claims in foreign counterparts of the foregoing for which an Authorization is required. The running royalty component of licenses may be purchased from Applied Biosystems or obtained by using Authorized reagents purchased from Authorized suppliers in accordance with the label rights accompanying such reagents.

2 Purchase of this instrument does not itself convey to the purchaser a complete license or right to perform the above processes. This instrument is also licensed under patents and apparatus and system claims in foreign counterparts thereof. No rights are granted expressly, by implication or by estoppel under composition claims or under other process or system claims owned or licensable by Applied Biosystems . For more information regarding licenses, please contact the Director of Licensing at Applied Biosystems , 850 Lin- coln Centre Drive, Foster City, California 94404. ABI Prism and the ABI Prism design, Applied Biosystems , GeneScan, Genotyper, MicroAmp, SeqEd, Sequence Navigator, and StockMarks are registered trademarks of Applied Biosystems or its subsidiaries in the and certain other countries. ABI, AmpFlSTR, BigDye, CATALYST, and GeneAssist are trademarks of Applied Biosysstems or its subsidiaries in the and certain other countries.

3 AmpliTaq, AmpliTaq Gold, GeneAmp and TaqMan are registered trademarks of Roche Molecular Systems, Inc. All other trademarks are the sole property of their respective owners. P/N 903565E. Technical Support .. xvii Contacting Technical Support .. xvii To Contact Technical Support by E-Mail .. xvii Hours for Telephone Technical Support .. xvii To Contact Technical Support by Telephone or Fax .. xvii To Reach Technical Support Through the Internet ..xx To Obtain Documents on Demand..xx Safety Information .. xxi 1 How the ABI PRISM 310 Genetic Analyzer Works .. 1-1. Introduction .. 1-1. In This Chapter .. 1-1. ABI Prism 310 Genetic Analyzer .. 1-1. Introduction .. 1-1. What the 310 Genetic Analyzer Does .. 1-2. DNA Sequencing .. 1-2. Required Software Files .. 1-2. GeneScan Software .. 1-3. Instrument Hardware: Front .. 1-5. Front of Instrument .. 1-5. Led Status Indicators.

4 1-5. Doors .. 1-5. Instrument Hardware: Back .. 1-6. Back of Instrument .. 1-6. On/Off Switch .. 1-6. Service LEDs .. 1-6. Communications Reset Button .. 1-7. Serial Connections .. 1-7. Power Cord Receptacle .. 1-7. Instrument Hardware: Behind the Doors .. 1-8. Overview .. 1-8. Instrument with Door Open .. 1-8. Autosampler Region Diagram .. 1-9. Autosampler Region Diagram Described .. 1-9. Gel Block Region Diagram .. 1-10. Gel Block Region Described .. 1-10. Detection Region Diagram .. 1-11. Detection Region Described .. 1-12. About the Macintosh Computer .. 1-13. Overview .. 1-13. System Requirements .. 1-13. RAM Requirements .. 1-13. iii Principles of Operation .. 1-14. Sample Labeling .. 1-14. Automated Sample Handling .. 1-14. Electrokinetic Sample Injection .. 1-14. Electrophoresis .. 1-14. Sample Fluorescence .. 1-14. Detection .. 1-15. Virtual Filters, Matrix Files and Dye Sets.

5 1-15. Compatibility of Dyes and Virtual Filters.. 1-15. Color Display of Data .. 1-16. Color Guide for Sequencing Raw Data .. 1-16. Color Guide for GeneScan Applications.. 1-17. Software for Data Collection and Analysis .. 1-18. Overview.. 1-18. Software Used with the Genetic Analyzer .. 1-18. Data Collection Software .. 1-18. Data Analysis Software.. 1-18. Other Software Provided with Your Computer .. 1-19. Overview.. 1-19. Consumables .. 1-19. Overview.. 1-19. Shared Consumables .. 1-20. About Capillaries .. 1-20. Removing Capillaries from the Packaging .. 1-20. Capillary Life .. 1-20. Capillary Types, Labels and Part Numbers.. 1-20. About Septa Strips for the 96-Well Tray.. 1-20. Using Septa Strips.. 1-21. Other Shared Consumables.. 1-23. Sequencing Consumables .. 1-24. Sequencing Polymer .. 1-24. Sequencing Application Kits.. 1-24. Sequencing Consumables Part Numbers.

6 1-24. GeneScan Consumables.. 1-25. GeneScan Polymer .. 1-25. GeneScan Application Kits .. 1-25. GeneScan Size Standard Kits .. 1-25. GeneScan Consumables Part Numbers.. 1-25. 2 Running Sequencing Experiments .. 2-1. Introduction .. 2-1. In This Chapter .. 2-1. iv About Running Sequencing Experiments .. 2-2. About this Chapter.. 2-2. About Cycle Sequencing .. 2-2. About DNA Sequencing .. 2-3. Description of Chemistry Table Levels .. 2-3. 310 Chemistry Options Table .. 2-4. About Cycle Sequencing Using Terminators.. 2-5. Summary of Procedures.. 2-5. Materials Required.. 2-6. 96-Well Sample Tray Accessories .. 2-8. 48-Well Sample Tray and Accessories.. 2-8. Software Required .. 2-8. How to Switch Between DNA Sequencing and the GeneScan Analysis Software .. 2-9. Switching Between Applications.. 2-9. How to Set Up the Reagents.. 2-10. Preparing Reagents .. 2-10. Storing the Buffer.

7 2-10. When to Replace Reagents .. 2-10. Preparing the 310 Genetic Analyzer for a DNA Sequencing Run With POP-6 .. 2-11. About These Procedures .. 2-11. Cleaning the Pump Block .. 2-11. Cleaning and Preparing the Syringe for Use .. 2-12. Loading Polymer .. 2-13. Setting Syringe Max Travel .. 2-13. Filling the Pump Block .. 2-14. Cleaning the Electrode.. 2-15. Installing the Capillary .. 2-15. Calibrating the Autosampler .. 2-16. Loading Buffers .. 2-17. Preheating the Heat Plate.. 2-17. DNA Template Preparation .. 2-18. Overview .. 2-18. Single-stranded DNA Templates .. 2-18. Plasmid DNA Templates .. 2-18. BAC DNA Templates.. 2-18. PCR Templates .. 2-19. DNA Template Quality.. 2-20. Using Control DNA .. 2-20. Poor Template Preparation.. 2-20. Contamination .. 2-20. Determining DNA Quality.. 2-21. Cleaning Up Dirty Templates .. 2-21. DNA Template Quantity.. 2-22. v Quantitation.

8 2-22. Amount to Use .. 2-22. Primer Design and Quantitation .. 2-23. Overview.. 2-23. Primer Design .. 2-23. Quantitation.. 2-23. Primer Problems and Possible Causes .. 2-24. Preparing Sequencing Reactions .. 2-25. Reagent Age and Reaction Storage.. 2-25. Reaction Tubes .. 2-25. Thermal Cyclers .. 2-25. BigDye Terminators .. 2-25. Cycle Sequencing .. 2-28. Overview.. 2-28. BigDye Terminators .. 2-28. Cycle Sequencing on the Catalyst 800 .. 2-30. Cycle Sequencing on the ABI PRISM 877 ITC .. 2-30. Preparing Extension Products for Electrophoresis .. 2-31. Overview.. 2-31. Spin Column Purification .. 2-31. Isopropanol Precipitation .. 2-33. Ethanol Precipitation.. 2-35. Ethanol/Sodium Acetate Precipitation .. 2-37. Denaturing and Loading Samples .. 2-40. About TSR and Samples in TSR.. 2-40. Preparing the TSR-Sample Mix .. 2-40. Minimum Sample Volume .. 2-40. Preparing a Portion of a Reaction Mixture for Analysis.

9 2-41. Loading the Samples.. 2-41. About Run Modules, Dye Set/Primer, and Matrix Files .. 2-42. Overview.. 2-42. Run Modules.. 2-42. Dye Set/Primer Files .. 2-42. Reference Table for Run Modules and Dye Set/Primer Files .. 2-43. Matrix Files.. 2-44. Preparing the Matrix Standards .. 2-45. Preparing Matrix Standard Samples .. 2-45. About dRhodamine Matrix Standards .. 2-45. Denaturing the Matrix Standard Samples .. 2-45. Preparing the Sample Standard .. 2-46. About Sample Standards.. 2-46. Preparing a TSR-Sample Standard Mix .. 2-46. Preparing the Sample Sheet and Injection List .. 2-47. vi Purpose .. 2-47. Preparing the Sample Sheet .. 2-47. Using a Previously Created Sample Sheet.. 2-48. Preparing an Injection List.. 2-49. Making Multiple Injections from One Sample Tube .. 2-50. Modifying an Injection List .. 2-51. Changing the Order of the Samples in a Run .. 2-52. Running and Monitoring Samples.

10 2-53. Testing the Capillary Window .. 2-53. Starting the Run .. 2-53. Monitoring the Run .. 2-53. Creating the DNA Sequencing Matrix File from the Matrix Standards.. 2-55. Overview .. 2-55. Verifying the Raw Data for the Matrix Standards Run .. 2-55. Generating a Matrix File from Matrix Standards .. 2-55. Checking Matrix Quality .. 2-60. Creating a DNA Sequencing Matrix from a Sample .. 2-63. Overview .. 2-63. Making Copies of the Sample File for the Sample Standard.. 2-63. Verifying Raw Data for the Sample Standard Matrix Run .. 2-63. Generating the Matrix File Using a Sample .. 2-64. Checking Matrix Quality .. 2-65. Analyzing The Data .. 2-65. Overview .. 2-65. Preparing the Syringes for a DNA Sequencing Run with DNA Sequencing Polymer (DSP) . 2-66. Overview .. 2-66. Cleaning and Preparing the Syringe for Use .. 2-66. Methods of Filling the Glass Syringe .. 2-66. Filling the Glass Syringe Manually.