Daniel:Notebook/HiResChrPaint/2014-7-8
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Strand Control
Andrew has pointed out to me that I should always do the processing on the probes, just to keep results consistent. I am fairly confident this is not the cause of my difficulties, but it is still wise to keep that particular aspect consistent. As such, I will be running through the more rigorous method of probe prep.
qPCR
I'll be running through V6S2 and V6S3, my favorites.
- Reaction Table
10 nM OligoPool | 2X Kapa SYBR qPCR MM | 100 uM AP1V61U | 100 uM AP2V6 | H20 | Total Volume | |
per rxn | 0.4 | 50 | 0.4 | 0.4 | 48.8 | 100 |
V6S2 (12.2x) | 4.88 | 610 | 4.88 | 4.88 | 595.36 | 1220 |
V6S3 (12.2x) | 4.88 | 610 | 4.88 | 4.88 | 595.36 | 1220 |
- Aliquot 100 uL into each of 12 qPCR tubes
- PCR protocol:
- PCR 3 min 95C
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto ii x17
- 72C 2 min
- 16C hold
- Pool 3 samples together
- Column Purification, elute with 40 uL EB
- Measure concentrations in nanodrop