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==Hybridization preparation and setup== # Add 100μl of 1mg/ml COT DNA and 1μg of amplified sample library to a new 1.5ml tube. # Add 10μl of each 100μM PE-HE1 and PE-HE2 Oligos to the amplified sample library plus COT DNA. (PE-HE1=Solexa_up, PE-HE2=Solexa_lo) # Close the tube’s lid and make a hole in the top of the tube’s cap with an 18 - 20 gauge or smaller needle. (Note: The hole in the top of the tube’s cap is a precaution to suppress contamination in the SpeedVac.) # Dry the amplified sample library/COT DNA/PE-HE Oligos in a SpeedVac on high heat (60°C). (Note: This step may take 30 minutes or longer. To minimize drying time, dry COT DNA ahead of time and then add the amplified sample library and PE- HE Oligos. Denaturation of the DNA with high heat is not problematic after linker ligation because the hybridization utilizes single-stranded DNA.) # To each dried-down amplified sample library/COT DNA/PE-HE Oligos, add 7.5μl of 2X SC Hybridization Buffer and 3μl of SC Hybridization Component A # Cover the hole in the tube’s cap with a sticker or small piece of laboratory tape. Vortex the amplified sample library/COT DNA/PE-HE Oligos plus Hybridization Cocktail (2X SC Hybridization Buffer + SC Hybridization Component A) for 10 seconds and centrifuge at maximum speed for 10 seconds. # Place the amplified sample library/COT DNA/PE-HE Oligos/Hybridization Cocktail in a 95°C heat block for 10 minutes to denature the DNA. # Centrifuge the amplified sample library/COT DNA/PE-HE/Hybridization Cocktail at maximum speed for 10 seconds at room temperature. # Transfer the amplified sample library/COT DNA/PE-HE Oligos/Hybridization Cocktail to the 4.5μl aliquot of Exome Library in a 0.2ml PCR tube prepared in Chapter 2 (entire volume can also be transferred to a 96-well PCR plate). Vortex for 3 seconds and centrifuge at maximum speed for 10 seconds. # Incubate in a thermocycler (with heated lid turned on) at 47°C for 72 hours.
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