Mapping 250K/500K SNP assay

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iv GeneChip® <strong>Mapping</strong> <strong>500K</strong> Assay Manual CHAPTER 4 96-Well Plate Protocol 33 96-WELL PLATE PROTOCOL 35 About This Protocol 35 BEFORE YOU BEGIN 38 Master Mix Preparation 38 Reagent Handling and Storage 38 Preparing the Work Area for Each Stage 39 thermal Cyclers, Plates and Plate Seals 40 PRogram Your Thermal Cyclers 41 Cutting Strip Tubes From Plates 41 STAGE 1: GENOMIC DNA PLATE PREPARATION 42 About this Stage 42 Location and Duration 42 Input Required 42 Equipment and Consumables Required 43 Reagents Required 44 Preparing the Genomic DNA Plate 44 Aliquoting Prepared Genomic DNA 45 What You Can Do Next 45 STAGE 2: RESTRICTION ENZYME DIGESTION 46 About this Stage 46 Location and Duration 46 Input Required From Previous Stage 46 Equipment and Consumables Required 46 Reagents Required 48 Important Information About This Stage 48 Prepare the Reagents, Equipment and Consumables 49 Prepare the Digestion Master Mix 50 Add Digestion Master Mix to Samples 51 STAGE 3: LIGATION 53 About this Stage 53 Location and Duration 53 Input Required From Previous Stage 53
contents v Equipment and Consumables Required 54 Reagents Required 55 Important Information About This Procedure 55 Prepare the Reagents, Consumables and Other Components 55 Prepare the Ligation Master Mix 57 Add Ligation Master Mix to Reactions 58 Dilute the Samples 59 What You Can Do Next 60 STAGE 4: PCR 61 About this Stage 61 Location and Duration 61 Input Required from Previous Stage 61 Equipment and Materials Required 62 Reagents Required 63 Gels and Related Materials Required 64 Important Information About This Stage 64 Prepare the Reagents, Consumables and Other Components 65 Add DNA to the Reaction Plates 66 Prepare the PCR Master Mix 68 Add PCR Master Mix to Samples 69 Load PCR Plates Onto Thermal Cyclers 70 Running Gels 72 What You Can Do Next 73 STAGE 5: PCR PRODUCT PURIFICATION AND ELUTION 74 About this Stage 74 Location and Duration 74 Input Required from Previous Stage 74 Equipment and Consumables Required 75 Reagents Required 76 Important Information About This Stage 76 Prepare the Reagents, Consumables and Other Components 77 Add Diluted EDTA to the PCR Products 78 Prepare the Clean-Up Plate 78 Pool the PCR Products 79 Purify the PCR Products 80 Elute The PCR Products 82 What You Can Do Next 83 STAGE 6: QUANTITATION AND NORMALIZATION 84 About this Stage 84 Location and Duration 84
Page 1 and 2: GeneChip ® Mapping 500K Assay Manu
Page 3: Contents CHAPTER 1 Overview 1 ABOUT
Page 7 and 8: contents vii Reagents Required 118
Page 9 and 10: contents ix CHAPTER 8 Troubleshooti
Page 11 and 12: contents xi STEP 4: PCR 257 Reagent
Page 13 and 14: 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
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EQUIPMENT AND CONSUMABLES REQUIRED
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ALIQUOTING PREPARED GENOMIC DNA To
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chapter 4 | 96-Well Plate Protocol
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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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POOL THE PCR PRODUCTS chapter 4 | 9
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Figure 4.5 Loading Optical Plates w
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Process Control Metrics Evaluate th
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Table 4.24 PROBLEM: Average Sample
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IMPORTANT INFORMATION ABOUT THIS ST
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Figure 4.6 Work Area Set Up for Sta
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Stage 8: Labeling ABOUT THIS STAGE
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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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