Daniel:Notebook/HiResChrPaint/2013-4-15: Difference between revisions
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|- style="font-size:12pt" align="center" | |- style="font-size:12pt" align="center" | ||
| height="15" | V4S2 | | height="15" | V4S2 | ||
| align=" | | align="center" | 12.5 | ||
| align=" | | align="center" | 12500 | ||
| align=" | | align="center" | 0.5 | ||
| align=" | | align="center" | 1.5 | ||
| align=" | | align="center" | 3.2 | ||
|- style="font-size:12pt" align="center" | |- style="font-size:12pt" align="center" | ||
| height="15" | V6S2 | | height="15" | V6S2 | ||
| align=" | | align="center" | 7.1 | ||
| align=" | | align="center" | 7100 | ||
| align=" | | align="center" | 0.3 | ||
| align=" | | align="center" | 0.5 | ||
| align=" | | align="center" | 1.9 | ||
|} | |} | ||
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Latest revision as of 22:01, 17 April 2013
Dye Coupling Test and Gel Quantification[edit]
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[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 so I only had to use one dye (Alexa 546)
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
Urea Gel Quantification[edit]
Set up samples according to following table
Sample | Low Mass Ladder | V4S2 w/Probe | V6S2 w/Probe | V4S2 w/o Probe | V6S2 w/o Probe | V6S1 w/o Probe |
uL sample | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 |
uL TBE | 5.5 | 5.5 | 5.5 | 5.5 | 5.5 | 5.5 |
uL 2X buffer | 6 | 6 | 6 | 6 | 6 | 6 |
Preran gel for ~45 minutes at 250 V Mixed samples in 0.2 mL eppendorf tubes Denatured samples for 10 minutes at 65 C immediately before loading Loaded samples according to following table
Sample | Low Mass Ladder | V4S2 w/Probe | V6S2 w/Probe | V4S2 w/o Probe | V6S2 w/o Probe | V6S1 w/o Probe |
Lane | 3 | 4 | 5 | 6 | 7 | 8 |
Ran gel for 20 minutes at 250 V Added 3 uL of SYBR Gold, incubated for 30 minutes on shaker Imaged using gel doc
Gel Quantification Results[edit]
Raw xls data
Sample | V4S2P | V6S2P | V4S2 | V6S2 | V6S1 |
ng/uL | 12.5 | 7.1 | 32.1 | 16.7 | 11.2 |
Sample | ng/uL | pg/uL | pmol/uL | uM Probe | Probes/oligo |
V4S2 | 12.5 | 12500 | 0.5 | 1.5 | 3.2 |
V6S2 | 7.1 | 7100 | 0.3 | 0.5 | 1.9 |