Mapping 250K/500K SNP assay

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114 GeneChip ® Mapping 500K Assay Manual Stage 9: Target Hybridization ABOUT THIS STAGE During this stage, each sample is loaded onto either a GeneChip® Human Mapping 250K Sty Array or a 250K Nsp Array. Three methods for performing this stage are presented. • Method 1 — Using a GeneAmp® PCR System 9700 Requires the use of a GeneAmp® PCR System 9700 located adjacent to the hybridization ovens. Samples are on a 96-well reaction plate. See Method 1 — Using a GeneAmp® PCR System 9700 on page 124. • Method 2 — Using an Applied Biosystems 2720 Thermal Cycler or an MJ Tetrad PTC-225 Thermal Cycler. Requires the use of an Applied Biosystems 2720 Thermal Cycler or an MJ Tetrad PTC-225 Thermal Cycler located adjacent to the hybridization ovens. Samples are on a 96-well reaction plate. See Method 2 — Using an Applied Biosystems 2720 Thermal Cycler or an MJ Tetrad PTC-225 Thermal Cycler on page 127. • Method 3 — Using Heat Blocks Requires the use of two heat blocks and Eppendorf tubes, one per sample. See Hybridizing Samples Using Heat Blocks on page 130. First, you will prepare a Hybridization Master Mix and add the mix to each sample. Then, based on the method you are using, you will denature the samples on a thermal cycler (methods 1 and 2) or on a heat block (method 3). After denaturation, you will load each sample onto the appropriate GeneChip Human Mapping 250K Array (Nsp or Sty) – one sample per array. The arrays are then placed into a hybridization oven that has been preheated to 49 °C. Samples are left to hybridize for 16 to 18 hours. Two operators are required for all of the methods.
LOCATION AND DURATION • Main Lab • Hands-on time: approximately 2 hours • Hybridization time: 16 to 18 hours INPUT REQUIRED FROM PREVIOUS STAGE The input required from Stage 8: Labeling is: Quantity Item 1 Plate of labeled DNA chapter 4 | 96-Well Plate Protocol 115 EQUIPMENT AND CONSUMABLES REQUIRED The following equipment and consumables are required for this stage. Refer to Appendix A, Reagents, Equipment, and Consumables Required for 96-Well Plate Protocol for vendor and part number information. Increased variability in Mapping 500K performance has been observed in GeneChip ® Hybridization Oven 640 models (P/N 800138 or 800189) manufactured prior to 2001. Check the serial number of your hybridization oven(s). If the serial numbers are 11214 or lower, contact Affymetrix for an upgrade.
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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
Page 75 and 76: REAGENTS REQUIRED chapter 4 | 96-We
Page 77 and 78: About Controls chapter 4 | 96-Well
Page 79 and 80: B P1 P2 P3 Figure 4.2 Transferring
Page 81 and 82: Table 4.16 PCR Master Mix ADD PCR M
Page 83 and 84: chapter 4 | 96-Well Plate Protocol
Page 85 and 86: WHAT YOU CAN DO NEXT chapter 4 | 96
Page 87 and 88: EQUIPMENT AND CONSUMABLES REQUIRED
Page 91 and 92: POOL THE PCR PRODUCTS chapter 4 | 9
Page 101 and 102: Figure 4.5 Loading Optical Plates w
Page 103 and 104: Process Control Metrics Evaluate th
Page 105 and 106: Table 4.24 PROBLEM: Average Sample
Page 109 and 110: EQUIPMENT AND CONSUMABLES REQUIRED
Page 111 and 112: IMPORTANT INFORMATION ABOUT THIS ST
Page 113 and 114: Figure 4.6 Work Area Set Up for Sta
Page 119 and 120: Stage 8: Labeling ABOUT THIS STAGE
Page 125: WHAT YOU CAN DO NEXT chapter 4 | 96
Page 129 and 130: Hybridizing Samples Using Heat Bloc
Page 135 and 136: Table 4.37 Hybridization Master Mix
Page 137 and 138: Load the Samples onto Arrays chapte
Page 139 and 140: Figure 4.8 Applying Tough-Spots ®
Page 145 and 146: Chapter 5 Washing, Staining, and Sc
Page 147 and 148: Introduction This chapter contains
Page 149 and 150: Reagent Preparation Wash A: Non-Str
Page 151 and 152: Experiment and Fluidics Station Set
Page 153 and 154: chapter 5 | Washing, Staining, and
Page 161 and 162: HANDLING THE GENECHIP ® PROBE ARRA
Page 165 and 166: Chapter 6 Fluidics Station Care and
Page 167 and 168: 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
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Appendix C Low Throughput Protocol
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Introduction The Affymetrix GeneChi
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BEFORE YOU BEGIN appendix C | Low T
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STEP 2: Restriction Enzyme Digestio
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DIGESTION PROCEDURE PRE-PCR CLEAN A
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STEP 3: Ligation REAGENTS AND EQUIP
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PCR STAGING AREA appendix C | Low T
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STEP 4: PCR REAGENTS AND EQUIPMENT
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PCR PROCEDURE PRE-PCR CLEAN ROOM ap
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MAIN LAB appendix C | Low Throughpu
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STEP 5: PCR Purification and Elutio
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appendix C | Low Throughput Protoco
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STEP 7: Fragmentation REAGENTS AND
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FRAGMENTATION PROCEDURE 1. Pre-heat
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LABELING PROCEDURE MAIN LAB appendi
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STEP 9: Target Hybridization REAGEN
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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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