Mapping 250K/500K SNP assay

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86 GeneChip ® <strong>Mapping</strong> <strong>500K</strong> Assay Manual Table 4.22 Reagents Required for Stage 6: Quantitation and Normalization Quantity Reagent As needed RB Buffer (from Clontech DNA Amplification Clean-Up Kit) 75 mL AccuGENE® water, molecular biology-grade IMPORTANT INFORMATION ABOUT THIS STAGE To help ensure the best results, carefully read the information below before you begin this stage of the protocol. • Prepare three independent dilutions of each sample for accurate concentration measurement. Average the results for each individual sample before normalizing. • The sample in each well must be normalized to 2 µg/µL in RB Buffer (90 µg in 45 µL RB Buffer). Do NOT determine an average concentration to use for every well. • The amount of DNA added to the arrays has been optimized for the best performance. Since not all wells will contain the same amount of DNA after purification, the eluted PCR products must be carefully normalized to 2 µg/µL before continuing to Stage 7: Fragmentation. • Normalize samples using RB Buffer (not water) to maintain the correct pH for subsequent steps. • The accuracy of the OD measurement is critical. Carefully follow the steps below and be sure the OD measurement is within the quantitative linear range of the instrument (0.2 to 0.8 OD). • The spectrophotometer plate reader should be calibrated regularly to ensure correct readings. • This protocol has been optimized using a UV spectrophotometer plate reader for quantitation. NOTE: The NanoDrop® will give different quantitation results. This protocol has not been optimized for use with this instrument. In
chapter 4 | 96-Well Plate Protocol 87 addition, the NanoDrop quantifies a single sample at a time and is not amenable to 96-well plate processing. PREPARE THE REAGENTS, EQUIPMENT AND CONSUMABLES Turn on the Spectrophotometer Plate Reader Turn on the spectrophotometer now and allow it to warm for 10 minutes before use. Prepare Your Work Area To prepare the work area: 1. Place a double cooling chamber on ice. 2. Label the 96-well reaction plate Fragment as this plate will also be used for the next stage), and place on the cooling chamber. 3. Place the following on the bench top: • Optical plates • Solution basin • AccuGENE® water 4. Label each optical plate as follows: OP1, OP2, OP3, OP4. 5. Vortex the RB Buffer and place on the bench top. 6. Prepare the purified, eluted PCR product plate as follows: A. If the plate was frozen, allow it to thaw in a cooling chamber on ice. B. Vortex the center of the plate at high speed for 3 sec. C. Spin down the plate at 2000 rpm for 30 sec. D. Place the plate on the bench top.
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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
Page 47 and 48: ABOUT THIS PROTOCOL 96-Well Plate P
Page 49 and 50: chapter 4 | 96-Well Plate Protocol
Page 51 and 52: PREPARING THE WORK AREA FOR EACH ST
Page 53 and 54: PROGRAM YOUR THERMAL CYCLERS chapte
Page 55 and 56: EQUIPMENT AND CONSUMABLES REQUIRED
Page 57 and 58: ALIQUOTING PREPARED GENOMIC DNA To
Page 61 and 62: Negative Controls chapter 4 | 96-We
Page 63 and 64: Table 4.7 Nsp I Digestion Master Mi
Page 65 and 66: Stage 3: Ligation ABOUT THIS STAGE
Page 67 and 68: REAGENTS REQUIRED chapter 4 | 96-We
Page 71 and 72: DILUTE THE SAMPLES To dilute the sa
Page 73 and 74: Stage 4: PCR ABOUT THIS STAGE chapt
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 85 and 86: WHAT YOU CAN DO NEXT chapter 4 | 96
Page 91 and 92: POOL THE PCR PRODUCTS chapter 4 | 9
Page 97: 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 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 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
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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
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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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