Daniel:Notebook/HiResChrPaint/2013-4-15

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Revision as of 19:06, 15 April 2013 by >Djacobse
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Dye Coupling Test and Gel Quantification

 Purpose:  Test dye coupling reaction, specifically the purification step, to ensure proper dye-coupled DNA is being captured.
 Two samples chosen from Gel Quantification (4/10/13). 
 Oligos coupled to appropriate dye.

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 so I only had to use one dye (Alexa )
Probe Alexa Dye
V4S1 488
V4S2 546
V4S3 594
V4S4 647
V6S1 488
V6S2 546
V6S3 594
V6S4 647
 6. Incubate reaction in the dark for 1 hour
 7. Column purify using ssDNA kit from Zymo

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