Mapping 250K/500K SNP assay

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REAGENTS REQUIRED 76 GeneChip ® <strong>Mapping</strong> <strong>500K</strong> Assay Manual ** Use only the PCR plate, adhesive film and thermal cyclers listed in Table 4.1 on page 40. The following reagents 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. The amounts listed are sufficient to process one full 96-well reaction plate. Table 4.20 Reagents Required for Stage 5: PCR Product Purification and Elution Quantity Reagent 1 Clean-Up Plate (Clontech) 3 mL EDTA, diluted to 0.1M (working stock is 0.5 M, pH 8.0) 5 mL RB Buffer 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. • The working stock of EDTA must be diluted to 0.1 M before use. •The AccuGENE® water listed in Appendix A must be used for this stage. Using in-house ddH 2O is not acceptable and can negatively impact downstream stages, particularly Stage 7: Fragmentation. The fragmentation reaction is very sensitive to pH and metal ion contamination. • To avoid cross-contamination and the introduction of air bubbles, pipette very careful when pooling the three PCR reactions for each sample onto the Clontech Clean-Up Plate. • Maintain the vacuum at 600 mbar.
chapter 4 | 96-Well Plate Protocol 77 • The PCR reactions contain significant contaminants including EDTA. These contaminants can affect subsequent steps unless removed by washing. Therefore, be sure to perform three water washes. • After the third wash, the wells must be completely dry before eluting the samples with RB Buffer. Any extra water carried with the RB Buffer to the next stage can result in over-fragmentation. • Immediately upon removal from the manifold, blot the bottom of the plate and wipe the bottom of each well. Any remaining liquid will quickly seep back into the wells. PREPARE THE REAGENTS, CONSUMABLES AND OTHER COMPONENTS Prepare the PCR Product Plates To prepare the PCR Product Plates from the previous stage: 1. Place the three PCR product plates on the bench top in plate holders. If frozen, allow them to thaw to room temperature. 2. Once at room temperature, vortex the center of each plate at high speed for 3 sec. 3. Spin down each plate at 2000 rpm for 30 sec. Dilute the Working Solution of EDTA Dilute the working stock of EDTA to a concentration of 0.1 M. A higher concentration may interfere with downstream steps. Setup the Manifold To set up the manifold: 1. Connect the manifold and regulator to a suitable vacuum source able to maintain 600 mbar. 2. Place the waste tray inside the base of the manifold. Do not turn on the vacuum at this time.
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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
Page 37 and 38: Chapter 3 Genomic DNA General Requi
Page 39 and 40: Introduction This chapter describes
Page 41 and 42: Sources of Human Genomic DNA chapte
Page 43 and 44: chapter 3 | Genomic DNA General Req
Page 45 and 46: 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 87: 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 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
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Figure 4.8 Applying Tough-Spots ®
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chapter 4 | 96-Well Plate Protocol
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chapter 4 | 96-Well Plate Protocol
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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
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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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