Mapping 250K/500K SNP assay

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54 GeneChip ® <strong>Mapping</strong> <strong>500K</strong> Assay Manual 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. Table 4.9 Equipment and Consumables Required for Stage 3: Ligation Quantity Item 1 Cooler, chilled to –20 °C 1 Cooling chamber, double, chilled to 4 °C (do not freeze) 1 Ice bucket, filled with ice 1 Marker, fine point, permanent 1 Microcentrifuge 1 Pipette, single channel P100 1 Pipette, single channel P1000 1 Pipette, 12-channel P20 1 Pipette, 12-channel P200 As needed Pipette tips for pipettes listed above; full racks 1 Plate centrifuge 2 Plate seal** 1 Solution basin, 55 mL 1 Thermal cycler** 1 strip Tubes, strip, 12 per strip 1 Tube, Eppendorf 2.0 mL 1 Vortexer ** Use only the PCR plate, adhesive film and thermal cyclers listed in Table 4.1 on page 40.
REAGENTS REQUIRED chapter 4 | 96-Well Plate Protocol 55 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 4.10 Reagents Required for Stage 3: Ligation Quantity Reagent 1 vial T4 DNA Ligase (400 U/µL; NEB) 1 vial T4 DNA Ligase Buffer (10X) 1 vial Adaptor, Nsp or Sty as appropriate (50 µM) 10 mL AccuGENE® water, molecular biology-grade IMPORTANT INFORMATION ABOUT THIS PROCEDURE To help ensure the best results, carefully read the information below before you begin this stage of the protocol. • Aliquot the T4 DNA Ligase Buffer (10X) after thawing for the first time to avoid multiple freeze-thaw cycles. See vendor instructions. • Be sure to use the correct adaptor (Nsp or Sty). PREPARE THE REAGENTS, CONSUMABLES AND OTHER COMPONENTS Thaw the Reagents and Digestion Stage Plate To thaw the reagents and Digestion Stage Plate: 1. Allow the following reagents to thaw on ice: • Adaptor Nsp I or Sty I as appropriate • T4 DNA Ligase Buffer (10X) Takes approximately 20 minutes to thaw.
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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
Page 15 and 16: About This Manual This manual is a
Page 17 and 18: chapter 1 | Overview 5 LD patterns
Page 19 and 20: Figure 1.1 GeneChip ® Mapping Assa
Page 21 and 22: chapter 1 | Overview 9 Gilman, B.,
Page 23 and 24: chapter 1 | Overview 11 23. Schaid,
Page 25 and 26: chapter 1 | Overview 13 35. Hu, N.,
Page 27 and 28: chapter 1 | Overview 15 50. Bignell
Page 29 and 30: chapter 1 | Overview 17 64. Gabriel
Page 31 and 32: Chapter 2 Laboratory Setup
Page 33 and 34: Introduction to Laboratory Setup Pl
Page 35 and 36: 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: Stage 3: Ligation ABOUT THIS STAGE
Page 71 and 72: DILUTE THE SAMPLES To dilute the sa
Page 73 and 74: 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 85 and 86: WHAT YOU CAN DO NEXT chapter 4 | 96
Page 91 and 92: POOL THE PCR PRODUCTS chapter 4 | 9
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 111 and 112: IMPORTANT INFORMATION ABOUT THIS ST
Page 113 and 114: Figure 4.6 Work Area Set Up for Sta
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WHAT YOU CAN DO NEXT chapter 4 | 96
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Stage 8: Labeling ABOUT THIS STAGE
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REAGENTS REQUIRED chapter 4 | 96-We
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chapter 4 | 96-Well Plate Protocol
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WHAT YOU CAN DO NEXT chapter 4 | 96
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LOCATION AND DURATION • Main Lab
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Hybridizing Samples Using Heat Bloc
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Table 4.37 Hybridization Master Mix
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Load the Samples onto Arrays chapte
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Figure 4.8 Applying Tough-Spots ®
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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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