Mapping 250K/500K SNP assay

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Chapter 1
About This Manual This manual is a guide for technical personnel conducting GeneChip® Mapping 500K experiments in the laboratory. It contains protocols for sample preparation, 96-well plate sample processing, and low throughput sample processing. It also includes instructions for washing, staining, and scanning arrays and generating genotype calls, as well as extensive troubleshooting information. A description of each chapter follows. Chapter 1: Scientific Overview: A description of the overall concept behind the GeneChip Mapping assay, including the biochemical process, data generation, potential applications, and a list of references. Chapter 2: Laboratory Setup: An explanation of the appropriate laboratory configuration for running GeneChip Mapping 500K experiments including how to set up a workflow to minimize the possibility of carryover contamination. Chapter 3: Genomic DNA Preparation: The requirements for genomic DNA, with recommended sources and methods for purification and quantitation. Chapter 4: 96-Well Plate Protocol: Detailed, step-by-step protocol for processing a full 96-well plate of human genomic DNA samples. Chapter 5: Washing, Staining, and Scanning: Basic protocols for fluidics station and scanner operation. Chapter 6: Fluidics Station Maintenance Procedures: Describes a weekly fluidics station bleach protocol and a monthly fluidics station decontamination protocol. Chapter 7: Analysis Workflow and Guidelines for Assessing Sample and Data Quality: Describes the analysis workflow in GeneChip® Operating Software (GCOS) and GeneChip® Genotyping Analysis Software (GTYPE), and provides information for assessing data quality. Chapter 8: Troubleshooting: Additional guidelines for obtaining optimal assay results including troubleshooting tips. Appendix A: Reagents, Equipment, and Supplies Required for 96-Well Plate Protocol: Includes vendor and part number information for the equipment and reagents required to run the 96-Well Plate Protocol. 3
Page 1 and 2: GeneChip ® Mapping 500K Assay Manu
Page 3 and 4: Contents CHAPTER 1 Overview 1 ABOUT
Page 5 and 6: contents v Equipment and Consumable
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: Chapter 1 Overview
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 59 and 60: chapter 4 | 96-Well Plate Protocol
Page 61 and 62: Negative Controls chapter 4 | 96-We
Page 63 and 64: 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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chapter 4 | 96-Well Plate Protocol
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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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EQUIPMENT AND CONSUMABLES REQUIRED
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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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EQUIPMENT AND CONSUMABLES REQUIRED
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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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