Materials and Reagents

1. 96-well V-bottom plates, elution catch (Greiner Bio-One GmbH, catalog number: 651101 )
   
2. Greiner UV star transparent, 96-well (Greiner Bio-One GmbH, catalog number: 82050-788 )
   Note: Currently, it is “VWR International, catalog number: 82050-788”.
   
3. Multiplate 96-well unskirted PCR plate (Bio-Rad Laboratories, catalog number: MLP-9601 )
   
4. Multiscreen deep well plate (Merck Millipore Corporation, catalog number: MDRLN0410 )
   
5. 2.4 ml deep well storage plate/pooling plate (Greiner Bio-One GmbH, catalog number: 780280 )
   
6. 100 ml solution basin (VWR International, Labcor^TM, catalog number: 730-014 )
   
7. 55 ml solution basin (VWR International, Labcor^TM, catalog number: 730-004 )
   
8. 15 ml centrifuge tubes (VWR International, catalog number: 20171-020 )
   
9. 50 ml centrifuge tubes (VWR International, catalog number: 21008-178 )
   
10. 2.0 ml Eppendorf tubes (VWR International, catalog number: 20901-540 )
    
11. 0.2 ml 12-strip tubes (VWR International, catalog number: 21008-940 )
    
12. Lab tape
    
13. Tough-Spots^®
    
14. Microseal ‘B’ adhesive seal (Bio-Rad Laboratories, model: MSB1001 )
    
15. MicroAmp^® clear adhesive film applied (Biosystems, catalog number: 4306311 )
    Note: Currently, it is “Thermo Fisher Scientific, Applied Biosystems^TM, catalog number: 4306311”.
    
16. Kimwipes (source as desired)
    
17. Pipet tips as appropriate for pipettes (source as desired)
    
18. AccuGENE^® water, molecular biology-grade (Affymetrix, catalog number: 71786 )
    
19. GenElute Mammalian Genomic DNA Miniprep (Sigma-Aldrich, catalog number: G1N70 )
    
20. Affymetrix SNP 6 Core Reagent Kit, 100 reactions (Affymetrix, catalog number: 901706 )
    
    1. 10x Sty I buffer/NE buffer 3 (New England Biolabs)
       
    2. T4 DNA Ligase (400 U/μl) (New England Biolabs)
       
    3. T4 DNA Ligase Buffer (10x) (New England Biolabs)
       
    4. Adaptor, sty (50 μM)
       
    5. PCR Primer 002 (100 μM)
       
    6. 10x Nsp I buffer/NE buffer 2 (New England Biolabs)
       
    7. NspI (10 U/μl) (New England Biolabs)
       
    8. Adaptor, nsp (50 μM)
       
    9. Elution buffer (Buffer EB)
       
    10. Fragmentation buffer (10x)
        
    11. Fragmentation reagent (DNase I)
        
    12. DNA Labeling reagent
        
    13. Terminal deoxynucleotidyl transferase (TdT) (30 U/μl)
        
    14. Terminal deoxynucleotidyl transferase buffer (TdT buffer) (5x)
        
    15. Oligo control reagent (OCR), 0100
        
    16. 10 mg/ml BSA (100x)
        
    17. Hybridization Master Mix
21. DNA marker (Bionexus, catalog number: BN2050 )
    
22. 2% TBE gel (Thermo Fisher Scientific, Invitrogen^TM, catalog number: G8008-02 )
    
23. Gel loading solution (Faster Better Media, catalog number: SB5N-8 )
    
24. Ethanol (Sigma-Aldrich, catalog number: 459844 )
    
25. 50 ml magnetic beads (AMPure, catalog number: A63881 )
    Note: It is named “Agencourt AMPure XP-PCR Purification” on Beckman Coulter website.
    
26. 4% TBE gel (Thermo Fisher Scientific, Invitrogen^TM, catalog number: G8008-04 )
    
27. MES hydrate (Sigma-Aldrich, catalog number: M8250 )
    
28. MES sodium salt (Sigma-Aldrich, catalog number: M3885 )
    Note: All catalog numbers are listed in the Affymetrix datasheet (http://media.affymetrix.com/support/downloads/manuals/genomewidesnp6_manual.pdf).
    
29. Clontech TITANIUM^TM DNA Amplification Kit (Clontech, catalog number: 639240 ) (see Recipes)
    
30. 12x MES Stock Solution (see Recipes)
    

Equipment

1. Applied Biosystems units
   
   1. 2720 Thermal Cycler
      
   2. GeneAmp^® PCR System 9700
      
   3. GeneAmp^® PCR System 9700 (silver block or gold-plated silver block)
2. 48 Genome-Wide Human SNP Array 6.0 (one array per sample)
   
3. GeneChip^® Hybridization Oven 640
   
4. Jitterbug
   
5. Spectrophotometer plate reader
   
6. Vacuum Manifold (Merck Millipore Corporation)
   
7. Vortexer
   
8. Cooler (chilled to -20 °C)
   
9. Cooling chamber (chilled to 4 °C) (do not freeze)
   
10. Cooling chamber (double, chilled to 4 °C) (do not freeze)
    
11. Ice bucket, filled with ice
    
12. Marker, fine point, permanent
    
13. Microcentrifuge
    
14. Pipet, 12-channel P1200
    
15. Pipet, 12-channel P200
    
16. Pipet, 12-channel P20 (accurate to within ± 5%)
    
17. Pipet, serological
    
18. Pipet, single channel P1000
    
19. Pipet, single channel P200
    
20. Plate centrifuge with deep-well capacity (54 mmH x 160 g)
    
21. Optical plates
    
22. Centrifuge plate holders
    Notes:
    
    1. Use only the PCR plate, adhesive film and thermal cyclers listed here.
       
    2. All relevant equipment numbers are listed in the Affymetrix datasheet.
    (http://media.affymetrix.com/support/downloads/manuals/genomewidesnp6_manual.pdf)
    

Procedure