Daniel:Notebook/HiResChrPaint/2013-4-15: Difference between revisions
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[[Image:ProbeQuant_041513.jpg]] | [[Image:ProbeQuant_041513.jpg]] | ||
{| | {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | ||
| align="center" | |- align="center" valign="bottom" | ||
| | | width="69" height="12" | Sample | ||
| | | width="69" | V4S2P | ||
| | | width="69" | V6S2P | ||
| | | width="69" | V4S2 | ||
| align="center" | | width="69" | V6S2 | ||
| | | width="69" | V6S1 | ||
| ng/uL||12. | |||
|- align="center" valign="bottom" | |||
| height="12" | ng/uL | |||
| align="right" | 12.5299723127746 | |||
| align="right" | 7.12098415157416 | |||
| align="right" | 32.1167808965069 | |||
| align="right" | 16.7206724376672 | |||
| align="right" | 11.2439322744476 | |||
|} | |} | ||
[[Daniel:Notebook/HiResChrPaint|Back to Calendar]] | [[Daniel:Notebook/HiResChrPaint|Back to Calendar]] |
Revision as of 00:42, 16 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 (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
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
Raw xls data
Sample | V4S2P | V6S2P | V4S2 | V6S2 | V6S1 |
ng/uL | 12.5299723127746 | 7.12098415157416 | 32.1167808965069 | 16.7206724376672 | 11.2439322744476 |