Daniel:Notebook/HiResChrPaint/2013-4-17
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Dye Coupling Test and EtOH Precipitation[edit]
Purpose: To test the dye coupling reaction (again), this time purifying post-reaction using ethanol precipitation. Will take longer (typical suggestion is overnight incubation) but could be more effective than the column preps.
Dye Coupling Reaction[edit]
1. Thaw out oligos, sodium bicarbonate solution, DMSO Use thermo device on Noi's desk to heat DMSO, sodium bicarb to 42 C 2. Add 2.5 uL DNA, 1.5 uL sodium bicarbonate to 0.2 mL eppendorf 3. Denature DNA by incubating at 95 C for 5 minutes, followed by snap cooling on ice 4. Thaw out dyes (they are in big -20C) and resuspend in 2.2 uL DMSO Use dyes immediately, they do not store 5. Add 1 uL of appropriate dye to oligo set (see below) Chose V4S2 and V6S2 because of high dna content Chose Alexa dye 546 to match typical color scheme of hi-res project 6. Incubate reaction in the dark for 1 hour
Ethanol Precipitation[edit]
1. Add: 0.5 uL NaOac (Recommended: 1/10X) 0.5 uL glycoblue (Recommended: 1/300X but that would be .03) and 30 uL 100% EtOH to each tube (Rec: 2.5X) 2. Transfer to a 1.5mL eppendorf and wrap in tinfoil to protect from light 3. Incubate overnight at -80C