Mapping 250K/500K SNP assay

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258 GeneChip ® Mapping 500K Assay Manual - 96-well plate: Bio-Rad, P/N MLP-9601 • 96-well Clear Adhesive Films: Applied Biosystems, P/N 4306311 • PCR Thermal Cycler (this assay has only been optimized for the following two thermal cyclers) 1 : - GeneAmp ® PCR System 9700 with gold-plated block, Applied Biosystems, or - DNA Engine Tetrad ® PTC-225, MJ Research 2 Program the thermal cycler in advance (see page 261) and switch on the thermal cycler 10 minutes before reactions are ready, so that the lid is heated. Make sure the ligated DNA from the ligation step was diluted to 100 µL with water. Prepare PCR Master Mix in Pre-PCR Clean room. Set up PCRs in PCR Staging Area. Prepare 3 PCRs for each sample and for each restriction enzyme (3 PCRs are required for each array). 1 The PCR process is covered by patents owned by Roche Molecular Systems, Inc. and F. Hoffmann-LaRoche Ltd (“Roche”). A license to use the PCR process for certain research and development activities accompanies the purchase of certain reagents from licensed suppliers when used in conjunction with an authorized thermal cycler. If you are using an MJ Research thermal cycler, your thermal cycler may not be an authorized thermal cycler. You should obtain authorization from Roche or ABI (see PCR licensing information in the MJ Research User Manual), if you are not already licensed. For information about obtaining a license contact The Director of Licensing at Applied Biosystems, 850 Lincoln Center Drive, Foster City, CA 94404 or the Licensing Department, Roche Molecular Systems, Inc., 1145 Atlantic Avenue, Alameda, CA 94501. 2 In 2004 MJ GeneWorks, Inc. and its subsidiary MJ Research, Inc. were purchased by Bio-Rad Laboratories, Inc. This model is no longer available. Bio-Rad has indicated that the DNA Engine Tetrad 2 gives similar performance with the same progams. Affymetrix has not tested the newer version.
PCR PROCEDURE PRE-PCR CLEAN ROOM appendix C | Low Throughput Protocol 259 The PCR reaction is sensitive to the concentration of primer used. It is critical that the correct amount of primer is added to the PCR reaction to achieve the correct distribution of fragments (200 to 1100 base pairs) in the products. Check the PCR reactions on a gel to ensure that the distribution is correct (see Figure C.2). 1. Prepare the following PCR Master Mix ON ICE (3 PCR reactions per sample) for Nsp I or Sty I ligation reactions and vortex at medium speed for 2 seconds (for multiple samples make a 5% excess): Table C.7 Stock Reagent 1 PCR 3 PCR Final Conc. in Sample H 2O 39.5 µL 118.5 µL Clontech TITANIUM Taq PCR Buffer (10X) 10 µL 30 µL 1X G-C Melt (5 M) 20 µL 60 µL 1 M dNTP (2.5 mM each) 14 µL 42 µL 350 µM (each) PCR Primer 002 (100 µM) 4.5 µL 13.5 µL 4.5 µM Clontech TITANIUM Taq DNA Polymerase (50X) 2 µL 6 µL 1X Total 90 µL 270 µL 90 µg of PCR product is needed for fragmentation.
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GeneChip ® Mapping 500K Assay Manu
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Contents CHAPTER 1 Overview 1 ABOUT
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contents v Equipment and Consumable
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contents vii Reagents Required 118
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contents ix CHAPTER 8 Troubleshooti
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contents xi STEP 4: PCR 257 Reagent
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Chapter 1 Overview
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About This Manual This manual is a
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chapter 1 | Overview 5 LD patterns
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Figure 1.1 GeneChip ® Mapping Assa
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chapter 1 | Overview 9 Gilman, B.,
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chapter 1 | Overview 11 23. Schaid,
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chapter 1 | Overview 13 35. Hu, N.,
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chapter 1 | Overview 15 50. Bignell
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chapter 1 | Overview 17 64. Gabriel
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Chapter 2 Laboratory Setup
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Introduction to Laboratory Setup Pl
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Main Lab Pre-PCR Clean Room chapter
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Chapter 3 Genomic DNA General Requi
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Introduction This chapter describes
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Sources of Human Genomic DNA chapte
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chapter 3 | Genomic DNA General Req
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Chapter 4 96-Well Plate Protocol
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ABOUT THIS PROTOCOL 96-Well Plate P
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chapter 4 | 96-Well Plate Protocol
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PREPARING THE WORK AREA FOR EACH ST
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PROGRAM YOUR THERMAL CYCLERS chapte
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EQUIPMENT AND CONSUMABLES REQUIRED
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ALIQUOTING PREPARED GENOMIC DNA To
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Negative Controls chapter 4 | 96-We
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Table 4.7 Nsp I Digestion Master Mi
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Stage 3: Ligation ABOUT THIS STAGE
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REAGENTS REQUIRED chapter 4 | 96-We
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DILUTE THE SAMPLES To dilute the sa
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Stage 4: PCR ABOUT THIS STAGE chapt
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About Controls chapter 4 | 96-Well
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B P1 P2 P3 Figure 4.2 Transferring
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Table 4.16 PCR Master Mix ADD PCR M
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WHAT YOU CAN DO NEXT chapter 4 | 96
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POOL THE PCR PRODUCTS chapter 4 | 9
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Figure 4.5 Loading Optical Plates w
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Process Control Metrics Evaluate th
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Table 4.24 PROBLEM: Average Sample
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IMPORTANT INFORMATION ABOUT THIS ST
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Figure 4.6 Work Area Set Up for Sta
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Stage 8: Labeling ABOUT THIS STAGE
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LOCATION AND DURATION • Main Lab
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Hybridizing Samples Using Heat Bloc
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Table 4.37 Hybridization Master Mix
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Load the Samples onto Arrays chapte
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Figure 4.8 Applying Tough-Spots ®
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Chapter 5 Washing, Staining, and Sc
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Introduction This chapter contains
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Reagent Preparation Wash A: Non-Str
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Experiment and Fluidics Station Set
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chapter 5 | Washing, Staining, and
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HANDLING THE GENECHIP ® PROBE ARRA
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Chapter 6 Fluidics Station Care and
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Introduction This chapter provides
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chapter 6 | Fluidics Station Care a
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White Clamp Figure 6.7 Releasing pe
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Figure 6.8 Troubleshooting decision
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PROBLEMS AND SOLUTIONS Table 6.3 Co
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Table 6.3 (Continued) Common error
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Table 6.4 (Continued) Common Error
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Table 6.5 (Continued) Other Problem
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Chapter 7 Analysis Workflow
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Introduction Software Requirements
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Figure 7.2 GTYPE main window with t
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Figure 7.3 Dynamic Model Mapping Al
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Table 7.1 Dynamic Model Mapping Alg
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FILE SETS chapter 7 | Analysis Work
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chapter 7 | Analysis Workflow 191 3
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NETAFFX SNP ANNOTATION chapter 7 |
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CALL RATE chapter 7 | Analysis Work
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chapter 7 | Analysis Workflow 197 G
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chapter 7 | Analysis Workflow 199 F
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chapter 7 | Analysis Workflow 201 S
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B2 OLIGO PERFORMANCE chapter 7 | An
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chapter 7 | Analysis Workflow 205 3
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Page 221 and 222: Chapter 8 Troubleshooting
Page 223 and 224: Assay Recommendations 211 Genotypin
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Page 227 and 228: Problem Likely Cause Solution Faint
Page 229 and 230: Problem Likely Cause Solution Posit
Page 231 and 232: Table 8.2 PROBLEM: Average Sample O
Page 233 and 234: When to Contact Technical Support c
Page 235 and 236: Appendix A Reagents, Equipment, and
Page 237 and 238: Reagents, Equipment, and Consumable
Page 239 and 240: appendix A | Reagents, Equipment, a
Page 245 and 246: Consumables Required GENECHIP ® AR
Page 247 and 248: Supplier Contact List Table A.10 Su
Page 249 and 250: Appendix B Thermal Cycler Programs
Page 251 and 252: ABOUT THIS APPENDIX 500K DIGEST 500
Page 253 and 254: 500K FRAGMENT 500K LABEL 500K HYB a
Page 255 and 256: Appendix C Low Throughput Protocol
Page 257 and 258: Introduction The Affymetrix GeneChi
Page 259 and 260: BEFORE YOU BEGIN appendix C | Low T
Page 261 and 262: STEP 2: Restriction Enzyme Digestio
Page 263 and 264: DIGESTION PROCEDURE PRE-PCR CLEAN A
Page 265 and 266: STEP 3: Ligation REAGENTS AND EQUIP
Page 267 and 268: PCR STAGING AREA appendix C | Low T
Page 269: STEP 4: PCR REAGENTS AND EQUIPMENT
Page 273 and 274: MAIN LAB appendix C | Low Throughpu
Page 275 and 276: STEP 5: PCR Purification and Elutio
Page 277 and 278: appendix C | Low Throughput Protoco
Page 279 and 280: STEP 7: Fragmentation REAGENTS AND
Page 281 and 282: FRAGMENTATION PROCEDURE 1. Pre-heat
Page 287 and 288: LABELING PROCEDURE MAIN LAB appendi
Page 289 and 290: STEP 9: Target Hybridization REAGEN
Page 291 and 292: HYBRIDIZATION PROCEDURE appendix C
Page 295 and 296: Appendix D Reagents, Instruments, a
Page 297 and 298: Appendix D
Page 299 and 300: appendix D | Reagents, Instruments,
Page 301 and 302: * Adaptor, Nsp 5' ATTATGAGCACGACAGA
Page 303 and 304: Table D.4 Reagents Supplied by Affy
Page 309 and 310: Fragmentation and Labeling appendix
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