Mapping 250K/500K SNP assay

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280 GeneChip ® Mapping 500K Assay Manual 2. Mix well. This Hybridization Cocktail Master Mix can be stored at –20°C before proceeding to the next step. 3. Transfer each of the labeled samples from the plate to a 1.5 mL Eppendorf tube. Aliquot 190 µL of the Hybridization Cocktail Master Mix into the 70 µL of labeled DNA samples as follows: Table C.15 Reagent Volume/Rx Labeled DNA 70 µL Hybridization Mix 190 µL Total 260 µL 4. Heat the 260 µL of hybridization mix and labeled DNA at 99°C in a heat block for exactly 10 minutes to denature. Denaturation of the labeled DNA sample is important to maximize binding to the oligonucleotides on the array surface. The sample must be heated to at least 95°C for 10 minutes prior to adding the DNA to the array. If a precipitate forms during this step, resuspend it before hybridization. Hybridize the arrays for at least 16 hours at 49°C. This temperature has been optimized for this product, and should be stringently followed. 5. Cool on crushed ice for 10 seconds. To avoid the formation of aggregates, do not leave on ice for longer than 10 seconds. 6. Spin briefly at 2,000 rpm in a microfuge to collect any condensate. 7. Place the tubes at 49°C for 1 minute.
appendix C | Low Throughput Protocol 281 8. Inject 200 µL denatured hybridization cocktail into the array. If there is anything that has come out of the solution, pipette briefly to resuspend before adding solution to the array. When processing multiple samples, leave samples at 49°C until ready to load onto the array. After injecting sample into an array, immediately place the array in the hybridization oven. 9. Hybridize at 49°C for 16 to 18 hours at 60 rpm. The remaining hybridization mix can be stored at –20°C for future use. The hybridization temperature is 49°C. This is different from the GeneChip ® expression assay, and the Mapping 10K and 100K assays.
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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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chapter 7 | Analysis Workflow 207 W
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Chapter 8 Troubleshooting
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Assay Recommendations 211 Genotypin
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chapter 8 | Troubleshooting 213 10.
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Problem Likely Cause Solution Faint
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Problem Likely Cause Solution Posit
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Table 8.2 PROBLEM: Average Sample O
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When to Contact Technical Support c
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Appendix A Reagents, Equipment, and
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Reagents, Equipment, and Consumable
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appendix A | Reagents, Equipment, a
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Page 243 and 244: 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 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
Page 291: 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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Mapping 250K/500K SNP assay

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