Mapping 250K/500K SNP assay

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232 GeneChip ® Mapping 500K 96-Well Plate Protocol THERMAL CYCLERS, PCR PLATES AND PLATE SEALS The 96-well plate protocol has been optimized using the following thermal cyclers, PCR plate and adhesive film. Use only the PCR plate, adhesive film and thermal cyclers listed in Table A.7. Using other PCR plates and film that are incompatible with the thermal cycler can result in crushed tubes, loss of sample, or poor results. Table A.7 Thermal Cyclers, PCR Plates and Plate Seals Optimized for Use With the Mapping 500K 96-Well Plate Protocol Area Thermal Cyclers Validated for Use Pre-PCR PCR and Post-PCR 2720 Thermal Cycler or GeneAmp ® PCR System 9700 by Applied Biosystems MJ Tetrad PTC-225 by Bio-Rad GeneAmp ® PCR System 9700 by Applied Biosystems (silver block or goldplated silver block) MJ Tetrad PTC-225 by Bio-Rad Plate Cover Multiplate 96-Well Unskirted PCR Plates Bio-Rad, P/N MLP-9601 Multiplate 96-Well Unskirted PCR Plates Bio-Rad, P/N MLP-9601 MicroAmp ® Clear Adhesive Films Applied Biosystems, P/N 4306311 MicroAmp ® Clear Adhesive Films Applied Biosystems, P/N 4306311
Consumables Required GENECHIP ® ARRAYS REQUIRED OTHER CONSUMABLES REQUIRED appendix A | Reagents, Equipment, and Consumables Required for 96-Well Plate Protocol 233 The following GeneChip® arrays are required: Table A.8 Arrays Required for the Mapping 500K 96-Well Plate Protocol Array Part Number GeneChip ® Human Mapping 250K Nsp Array* (minimum order is 200 arrays) 520330 (1 array) GeneChip ® Human Mapping 250K Nsp Array* 900768 (30 arrays) GeneChip ® Human Mapping 250K Sty Array* (minimum order is 200 arrays) 520331 (1 array) GeneChip ® Human Mapping 250K Sty Array* 900770 (30 arrays) * Denotes critical equipment. Vendors not listed here have not been tested and verified at Affymetrix. Table A.9 Other Consumables Required for the Mapping 500K 96-Well Plate Protocol Item Manufacturer/ Distributor Gel, Reliant ® Gel System, precast agarose gel (2% SeaKem Gold, TBE) Part Number Laboratory Location Cambrex 54939 Main Lab DNA Marker Bionexus All Purpose Hi-Lo, BN2050 Main Lab Gel Loading Solution Sigma-Aldrich G2526 Main Lab
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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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Page 197 and 198: Figure 7.3 Dynamic Model Mapping Al
Page 199 and 200: Table 7.1 Dynamic Model Mapping Alg
Page 201 and 202: FILE SETS chapter 7 | Analysis Work
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Page 205 and 206: NETAFFX SNP ANNOTATION chapter 7 |
Page 207 and 208: CALL RATE chapter 7 | Analysis Work
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Page 215 and 216: B2 OLIGO PERFORMANCE chapter 7 | An
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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
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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
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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 and 270: STEP 4: PCR REAGENTS AND EQUIPMENT
Page 271 and 272: PCR PROCEDURE PRE-PCR CLEAN ROOM ap
Page 273 and 274: MAIN LAB appendix C | Low Throughpu
Page 275 and 276: STEP 5: PCR Purification and Elutio
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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
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Appendix D Reagents, Instruments, a
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Appendix D
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appendix D | Reagents, Instruments,
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* Adaptor, Nsp 5' ATTATGAGCACGACAGA
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Table D.4 Reagents Supplied by Affy
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Fragmentation and Labeling appendix
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Mapping 250K/500K SNP assay

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