Mapping 250K/500K SNP assay

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50 GeneChip ® <strong>Mapping</strong> <strong>500K</strong> Assay Manual B. Pulse spin for 3 sec. C. Place in the cooling chamber. Preheat the Thermal Cycler Lid Power on the thermal cycler to preheat the lid. Leave the block at room temperature. PREPARE THE DIGESTION MASTER MIX The same team or individual operator should not process samples with both Nsp and Sty enzymes on the same day. Best practice is to process samples for either Nsp or Sty on a given day. Keeping all reagents and tubes on ice, prepare the Digestion Master Mix as follows: 1. To the 2.0 mL Eppendorf tube, add the appropriate volumes of the following reagents based on Table 4.7 (Nsp) or Table 4.8 (Sty): • AccuGENE® Water • NE Buffer • BSA 2. Remove the appropriate enzyme (Nsp I or Sty I) from the freezer and immediately place in a cooler. 3. Pulse spin the enzyme for 3 sec. 4. Immediately add the enzyme to the master mix, then place remaining enzyme back in the cooler. 5. Vortex the master mix at high speed 3 times, 1 sec each time. 6. Pulse spin for 3 sec. 7. Place in the cooling chamber. 8. Return any remaining enzyme to the freezer. 9. Proceed immediately to Add Digestion Master Mix to Samples on page 51.
Table 4.7 Nsp I Digestion Master Mix ADD DIGESTION MASTER MIX TO SAMPLES To add Digestion Master Mix to samples: chapter 4 | 96-Well Plate Protocol 51 Reagent 1 Sample 96 Samples (15% extra) AccuGENE® Water 11.55 µL 1275.1 µL NE Buffer 2 (10X) 2 µL 220.8 µL BSA (100X; 10 mg/mL) 0.2 µL 22.1 µL Nsp I (10 U/µL) 1 µL 110.4 µL Total 14.75 µL 1628.4 µL Table 4.8 Sty I Digestion Master Mix Reagent 1 Sample 96 Samples (15% extra) AccuGENE® Water 11.55 µL 1275.1 µL NE Buffer 3 (10X) 2 µL 220.8 µL BSA (100X; 10 mg/mL) 0.2 µL 22.1 µL Sty I (10 U/µL) 1 µL 110.4 µL Total 14.75 µL 1628.4 µL 1. Using a single channel P200 pipette, aliquot 135 µL of Digestion Master Mix to each tube of the strip tubes labeled Dig. 2. Using a 12-channel P20 pipette, add 14.75 µL of Digestion Master Mix to each DNA sample in the cooling chamber on ice. The total volume in each well is now 19.75 µL.
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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
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
Page 55 and 56: EQUIPMENT AND CONSUMABLES REQUIRED
Page 57 and 58: ALIQUOTING PREPARED GENOMIC DNA To
Page 61: Negative Controls chapter 4 | 96-We
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 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 111 and 112: IMPORTANT INFORMATION ABOUT THIS ST
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Figure 4.6 Work Area Set Up for Sta
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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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Stage 8: Labeling ABOUT THIS STAGE
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REAGENTS REQUIRED chapter 4 | 96-We
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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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