Mapping 250K/500K SNP assay

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OTHER REAGENTS REQUIRED 228 GeneChip ® Mapping 500K 96-Well Plate Protocol Table A.4 Other Reagents Required for the Mapping 500K 96-Well Plate Protocol Reagent Vendor Description Part Number TITANIUM DNA Amplification Kit DNA Amplification Clean- Up Kit TITANIUM Taq DNA Polymerase (50X) and TITANIUM Taq PCR Buffer Clontech Contains: 50X TITANIUM Taq DNA Polymerase 10X TITANIUM Taq PCR Buffer GC-Melt dNTPs 639240 (300 reactionsenough for 100 samples) Clontech Each kit includes RB Buffer 1 Plate: 636974 4 Plates: 636975 Clontech Conains: 50X Clontech TITANIUM Taq DNA Polymerase 10X Clontech TITANIUM Taq PCR Buffer P/N 639209 GC-Melt Clontech 5 M 639238 dNTPs * Takara mixture of dATP, dCTP, dGTP, dTTP at Fisher Scientific 2.5 mM each 4030 TAK 4030 Denhart’s Solution Sigma-Aldrich D2532 DMSO Sigma-Aldrich D5879 EDTA Ambion 0.5 M, pH 8.0 (dilute to 0.1 M before use) 9260G Herring Sperm DNA (HSDNA) Promega D1815 Human Cot-1 DNA® Invitrogen 15279-011 MES Hydrate SigmaUltra Sigma-Aldrich M5287 MES Sodium Salt Sigma-Aldrich M5057 Reduced EDTA TE Buffer TEKnova 10 mM Tris HCL, 0.1 mM EDTA, pH 8.0 T0223 RB Buffer Contained in the DNA Amplification Clean-Up Kit listed above. Tetramethyl Ammonium Chloride (TMACL; 5M) Sigma-Aldrich 5M T3411
appendix A | Reagents, Equipment, and Consumables Required for 96-Well Plate Protocol 229 Table A.4 Other Reagents Required for the Mapping 500K 96-Well Plate Protocol Reagent Vendor Description Part Number Tween-20, 10% Pierce 10%, diluted to 3% in molecular biologygrade water Water, AccuGENE ® Cambrex AccuGENE ® Molecular Biology-Grade Water, 1 L Equipment and Software Required The 96-well plate protocol has been optimized using the following equipment and software. AFFYMETRIX EQUIPMENT AND SOFTWARE REQUIRED 28320 (Surfact- AmpsQ®) 51200 * dNTPs from Invitrogen (P/N R72501) have been tested on a limited basis with similar results. You should test in your own lab prior to full scale production. Table A.5 Affymetrix Equipment and Software Required Item Part Number GeneChip ® Fluidics Station 450* 00-0079 GeneChip ® Hybridization Oven 640* 800139 GeneChip ® Scanner 3000 7G* 00-0205 GeneChip ® Operating Software version 1.4* 690031 GeneChip ® Genotyping Analysis Software 4.0* 690051 * Denotes critical reagents, equipment or supplies. Formulations or vendors not listed here have not been tested and verified at Affymetrix. In some cases, lower performance has been demonstrated by reagents from non-qualified vendors.
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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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Page 191 and 192: Chapter 7 Analysis Workflow
Page 193 and 194: Introduction Software Requirements
Page 195 and 196: Figure 7.2 GTYPE main window with t
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
Page 209 and 210: chapter 7 | Analysis Workflow 197 G
Page 211 and 212: chapter 7 | Analysis Workflow 199 F
Page 213 and 214: chapter 7 | Analysis Workflow 201 S
Page 215 and 216: B2 OLIGO PERFORMANCE chapter 7 | An
Page 217 and 218: chapter 7 | Analysis Workflow 205 3
Page 219 and 220: chapter 7 | Analysis Workflow 207 W
Page 221 and 222: Chapter 8 Troubleshooting
Page 223 and 224: Assay Recommendations 211 Genotypin
Page 225 and 226: chapter 8 | Troubleshooting 213 10.
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: appendix A | Reagents, Equipment, a
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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 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
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
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HYBRIDIZATION PROCEDURE appendix C
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appendix C | Low Throughput Protoco
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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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