Mapping 250K/500K SNP assay

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REAGENTS REQUIRED 98 GeneChip ® <strong>Mapping</strong> <strong>500K</strong> Assay Manual 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 4.28 Reagents Required for Stage 7: Fragmentation Quantity Reagent 1 vial Fragmentation Buffer (10X) 1 vial Fragmentation Reagent (DNase I) 2 mL AccuGENE® water, molecular biology-grade GELS AND RELATED MATERIALS REQUIRED The following gels and related materials 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 4.29 Quantity Reagent 5 4% TBE Gel 10 DNA Markers, 5 µL each As needed Gel loading solution
IMPORTANT INFORMATION ABOUT THIS STAGE chapter 4 | 96-Well Plate Protocol 99 To help ensure the best results, carefully read the information below before you begin this stage of the protocol. • Purified PCR product must be normalized to 90 µg DNA in 45 µL RB Buffer. • The degree of fragmentation is critical. Perform this stage carefully to ensure uniform, reproducible fragmentation. • The Fragmentation Reagent is extremely temperature sensitive. It rapidly loses activity at higher temperatures. To avoid loss of activity: - Dilute the Fragmentation Reagent immediately prior to use. - Keep at –20 °C until ready to use. Transport and hold in a –20 °C cooler. Return to the cooler immediately after use. - Perform these steps rapidly and without interruption. • The Fragmentation Reagent (DNase I) may adhere to the walls of some microfuge tubes and 96-well plates. To ensure the accurate amount of DNase I in the fragmentation reaction (Stage 7: Fragmentation), the strip tubes used for this stage must be cut from Bio-Rad 96-well unskirted PCR plates, P/N MLP-9601. See Cutting Strip Tubes From Plates on page 41 and Table 4.1 on page 40 for more information on these plates. • The Fragmentation Reagent is viscous and requires extra care when pipetting. Follow these guidelines: - When aspirating, allow enough time for the correct volume of solution to enter the pipette tip. - Avoid excess solution on the outside of the pipette tip. • Be sure to use the AccuGENE® water listed in Appendix A. Using in-house ddH2O or other water can negatively affect your results. The reaction in Stage 7: Fragmentation is particularly sensitive to pH and metal ion contamination. • All additions, dilutions and mixing must be performed on ice.
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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
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ABOUT THIS PROTOCOL 96-Well Plate P
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chapter 4 | 96-Well Plate Protocol
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PREPARING THE WORK AREA FOR EACH ST
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PROGRAM YOUR THERMAL CYCLERS chapte
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EQUIPMENT AND CONSUMABLES REQUIRED
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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
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 and 88: 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: EQUIPMENT AND CONSUMABLES REQUIRED
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
Page 149 and 150: Reagent Preparation Wash A: Non-Str
Page 151 and 152: Experiment and Fluidics Station Set
Page 153 and 154: chapter 5 | Washing, Staining, and
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HANDLING THE GENECHIP ® PROBE ARRA
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chapter 5 | Washing, Staining, and
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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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