Daniel:Notebook/HiResChrPaint/2013-4-17

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Revision as of 20:44, 17 April 2013 by >Djacobse (Created page with "==Dye Coupling Test and Gel Quantification-EtOH Precipitation== Purpose: To test the dye coupling reaction (again), this time purifying post-reaction using ethanol precipita...")
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Dye Coupling Test and Gel Quantification-EtOH Precipitation

 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

 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
 7. Column purify using ssDNA kit from Zymo

Ethanol Precipitation

 1. Add 0.5 (1/10X) uL NaOac, 0.5 uL glycoblue (Recommended: 1/300X but that would be .03) and 30 uL 100% EtOH to each tube (Rec: 2.5X)
 2. Incubate overnight at -80C

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