Mapping 250K/500K SNP assay

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294 GeneChip ® Mapping 500K Assay Manual Table D.6 Reagents, Equipment and Supplies Not Supplied by Affymetrix Assay Step Reagents Equipment Ligation T4 DNA Ligase: New England Biolabs (NEB), P/N M0202L containing: T4 DNA Ligase Buffer: New England Biolabs (NEB), P/N B0202S Molecular Biology Grade Water: BioWhittaker Molecular Applications/Cambrex, P/N 51200 Adaptor Nsp (50 µM): Affymetrix, P/N 900596 for 30 Rxnx and P/N 900697 for 100 Rxns (if running Nsp Array) available in the GeneChip ® Mapping 250K Nsp Assay Kit: 30 Rxn Kit P/N 900766, 100 Rxn Kit P/N 900753 Adaptor Sty (50 µM): Affymetrix, P/N 900597 for 30 Rxns and P/N 900698 for 100 Rxns (if running Sty Array) available in the GeneChip ® Mapping 250K Sty Assay Kit: 30 Rxn Kit P/N 900765, 100 Rxn Kit P/N 900754 Thermal Cycler
appendix D | Reagents, Instruments, and Supplies Required for Low Throughput Protocol 295 Table D.6 Reagents, Equipment and Supplies Not Supplied by Affymetrix Assay Step Reagents Equipment PCR G-C Melt (5 M): Clontech, P/N 639238 H 2O (Molecular Grade Biology Water): BioWhittaker Molecular Applications/Cambrex, P/N 51200 dNTP (2.5 mM each): Takara, P/N 4030; Fisher Scientific, P/N TAK 4030; or Invitrogen, P/N R72501 † (FN1) PCR Primer, 002 (100 µM): Affymetrix, P/N 900595 for 30 Rxns and P/N 900702 for 100 Rxns (if running Nsp Array), available in the GeneChip ® Mapping 250K Nsp Assay Kits: 30 Rxn Kit P/N 900766 100 Rxn Kit P/N 900753 PCR Primer, 002 (100 µM): Affymetrix, P/N 900595 for 30 Rxns and P/N 900702 for 100 Rxns (if running Sty Array), available in the GeneChip ® Mapping 250K Sty Assay Kits: 30 Rxn Kit P/N 900765 100 Rxn Kit P/N 900754 Clontech TITANIUM Taq DNA Polymerase (50X): Clontech, P/N 639209 50X Clontech TITANIUM Taq DNA Polymerase 10X Clontech TITANIUM Taq PCR Buffer 2% TBE Gel:BMA Reliant precast (2% SeaKem ® Gold): Cambrex Bio Science, P/N 54939 All Purpose Hi-Lo DNA Marker: Bionexus, Inc., P/N BN2050, or Direct Load Wide Range DNA Marker: Sigma, P/N D7058 Gel Loading Solution: Sigma, P/N G2526 PCR Tubes*: (must be compatible and qualified with MJ DNA Engine Tetrad ®** , or ABI GeneAmp ® PCR System) For example: Individual tubes: Bio-Rad, P/N TWI-0201 8-Tube Strips, thin-wall (0.2 mL): Bio-Rad, P/N TBS-0201; Strip of 8 caps: Bio-Rad, P/N TCS-0801 Plate: (must be compatible and qualified with MJ DNA Engine Tetrad ®** , or ABI GeneAmp ® PCR System) 96-well plate: Bio-Rad, P/N MLP-9601 96-well PLT. Clear Adhesive Films: Applied Biosystems, P/N 4306311 PCR Thermal Cycler ‡ * (Ramp speeds on these models are critical - assay protocols have been optimized for these two thermal cycler models only): MJ Research DNA Engine Tetrad ®** (PTC-225) 96 well block, or ABI GeneAmp ® PCR System 9700 - gold plated 96 well block gel apparatus Jitterbug: Boekel Scientific, model 130000
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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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Consumables Required GENECHIP ® AR
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Supplier Contact List Table A.10 Su
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Appendix B Thermal Cycler Programs
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ABOUT THIS APPENDIX 500K DIGEST 500
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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 and 292: HYBRIDIZATION PROCEDURE appendix C
Page 295 and 296: Appendix D Reagents, Instruments, a
Page 297 and 298: Appendix D
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Page 301 and 302: * Adaptor, Nsp 5' ATTATGAGCACGACAGA
Page 303 and 304: Table D.4 Reagents Supplied by Affy
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Page 309 and 310: Fragmentation and Labeling appendix
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Mapping 250K/500K SNP assay

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