Daniel:Notebook/HiResChrPaint/2013-4-17: Difference between revisions
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>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 | ==Dye Coupling Test and 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. | 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=== | ===Dye Coupling Reaction=== | ||
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Chose Alexa dye 546 to match typical color scheme of hi-res project | Chose Alexa dye 546 to match typical color scheme of hi-res project | ||
6. Incubate reaction in the dark for 1 hour | 6. Incubate reaction in the dark for 1 hour | ||
===Ethanol Precipitation=== | ===Ethanol Precipitation=== | ||
1. Add 0.5 (1/10X) | 1. Add: | ||
2. Incubate overnight at -80C | 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 | |||
[[Daniel:Notebook/HiResChrPaint|Back to Calendar]] | [[Daniel:Notebook/HiResChrPaint|Back to Calendar]] |
Latest revision as of 22:43, 17 April 2013
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