Daniel:Notebook/HiResChrPaint/2013-4-15: Difference between revisions
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Use thermo device on Noi's desk to heat DMSO, sodium bicarb to 42 C | 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 | 2. Add 2.5 uL DNA, 1.5 uL sodium bicarbonate to 0.2 mL eppendorf | ||
3. Thaw out dyes (they are in big -20C) and resuspend in 2.2 uL DMSO | 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 | 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 | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | ||
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6. Incubate reaction in the dark for 1 hour | |||
7. Column purify using ssDNA kit from Zymo | |||
[[Daniel:Notebook/HiResChrPaint|Back to Calendar]] | [[Daniel:Notebook/HiResChrPaint|Back to Calendar]] |
Revision as of 19:06, 15 April 2013
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
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