Mapping 250K/500K SNP assay

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142 GeneChip ® <strong>Mapping</strong> <strong>500K</strong> Assay Manual Probe Array Wash and Stain The Affymetrix staining protocol for mapping arrays is a three stage process consisting of a Streptavidin Phycoerythin (SAPE) stain, followed by an antibody amplification step and final stain with Streptavidin Phycoerythin (SAPE). Following staining, the array is filled with Array Holding Buffer prior to scanning as outlined in Table 5.5. 1. After 16 to 18 hours of hybridization, remove the hybridization cocktail from the probe array and set it aside in a microcentrifuge vial. Store on ice during the procedure or at –80°C for long-term storage. 2. Fill the probe array completely with 270 µL of Array Holding Buffer. If necessary, the probe array can be stored in the Array Holding Buffer at 4°C for up to 3 hours before proceeding with washing and staining. Equilibrate the probe array to room temperature before washing and staining.
chapter 5 | Washing, Staining, and Scanning Arrays 143 Preparing the Staining Reagents Prepare the following reagents. Volumes given are sufficient for one probe array. Mix well. Table 5.1 Stain Buffer Components 1X Final Concentration H 2O 800.04 µL SSPE (20X) 360 µL 6X Tween-20 (3%) 3.96 µL 0.01% Denhardt’s (50X) 24 µL 1X Subtotal 1188 µL Subtotal/2 594 µL SAPE Stain Solution Streptavidin Phycoerythrin (SAPE) should be stored in the dark at 4°C, either foil-wrapped or in an amber tube. Remove SAPE from refrigerator and tap the tube to mix well before preparing stain solution. Always prepare the SAPE stain solution immediately before use. Do not freeze either concentrated SAPE or diluted SAPE stain solution. Table 5.2 SAPE Solution Mix Components Volume Final Concentration Stain Buffer 594 µL 1X 1 mg/mL Streptavidin Phycoerythrin (SAPE) 6.0 µL 10 µg/mL Total 600 µL Mix well.
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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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EQUIPMENT AND CONSUMABLES REQUIRED
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POOL THE PCR PRODUCTS chapter 4 | 9
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WHAT YOU CAN DO NEXT chapter 4 | 96
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Figure 4.5 Loading Optical Plates w
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Page 105 and 106: Table 4.24 PROBLEM: Average Sample
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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 115 and 116: chapter 4 | 96-Well Plate Protocol
Page 119 and 120: Stage 8: Labeling ABOUT THIS STAGE
Page 121 and 122: REAGENTS REQUIRED chapter 4 | 96-We
Page 127 and 128: LOCATION AND DURATION • Main Lab
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: chapter 5 | Washing, Staining, and
Page 157 and 158: 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
Page 169 and 170: chapter 6 | Fluidics Station Care a
Page 179 and 180: White Clamp Figure 6.7 Releasing pe
Page 181 and 182: Figure 6.8 Troubleshooting decision
Page 183 and 184: PROBLEMS AND SOLUTIONS Table 6.3 Co
Page 185 and 186: Table 6.3 (Continued) Common error
Page 187 and 188: Table 6.4 (Continued) Common Error
Page 189 and 190: Table 6.5 (Continued) Other Problem
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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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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