Mapping 250K/500K SNP assay

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126 GeneChip ® <strong>Mapping</strong> <strong>500K</strong> Assay Manual Operator 2 Tasks 1. Cover the septa on each array with a Tough-Spot (Figure 4.8). 2. For every 4 arrays: A. Load the arrays into an oven tray evenly spaced. B. Immediately place the tray into the hybridization oven. Do not allow loaded arrays to sit at room temperature for more than approximately 1.5 minute. Ensure that the oven is balanced as the trays are loaded, and ensure that the trays are rotating at 60 rpm at all times. Because you are loading 4 arrays per tray, each hybridization oven will have a total of 32 arrays. Operators 1 and 2 • Load no more than 32 arrays in one hybridization oven at a time. • All 96 samples should be loaded within 1 hour. • Store the remaining samples and any samples not yet hybridized in a tightly sealed plate at -20 °C. • Allow the arrays to rotate at 49 °C, 60 rpm for 16 to 18 hours. Allow the arrays to rotate in the hybridization ovens for 16 to 18 hours at 49°C and 60 rpm. This temperature is optimized for this product, and should be stringently followed.
Figure 4.8 Applying Tough-Spots ® to the array cartridge chapter 4 | 96-Well Plate Protocol 127 Method 2 — Using an Applied Biosystems 2720 Thermal Cycler or an MJ Tetrad PTC-225 Thermal Cycler For this method, you can use an Applied Biosystems 2720 Thermal Cycler or an MJ Tetrad PTC-225 Thermal Cycler. The thermal cycler must be located adjacent to the hybridization ovens. Because the lids on these thermal cyclers do not slide back, you will process 24 samples at a time. Add Hybridization Master Mix and Denature To add Hybridization Master Mix and denature the samples: 1. Pour 20.9 mL Hybridization Master Mix into a solution basin. 2. Using a 12-channel P200 pipette, add 190 µL of Hybridization Master Mix to each sample on the Label Plate. Total volume in each well is 260 µL. 3. IMPORTANT Seal the plate tightly with adhesive film. 4. Vortex the center of the plate for 3 minutes. 5. Cut the plate into 4 strips of two rows each. 6. Put each strip of 24 samples into a plate holder, 2 strips per holder. 7. Spin down the strips at 2000 rpm for 30 sec.
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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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REAGENTS REQUIRED chapter 4 | 96-We
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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
Page 87 and 88: EQUIPMENT AND CONSUMABLES REQUIRED
Page 89 and 90: chapter 4 | 96-Well Plate Protocol
Page 91 and 92: POOL THE PCR PRODUCTS chapter 4 | 9
Page 95 and 96: WHAT YOU CAN DO NEXT chapter 4 | 96
Page 97 and 98: REAGENTS REQUIRED chapter 4 | 96-We
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 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: Load the Samples onto Arrays chapte
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
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
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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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