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
qPCR Results
Nanodrop Results
V6S2A | V6S2B | V6S2C | V6S2D | V6S3A | V6S3B | V6S3C | V6S3D | |
ng/uL | 99.9 | 95.4 | 94.6 | 84.5 | 110 | 112.2 | 111.6 | 99.8 |
ug in 40 uL | 4.0 | 3.8 | 3.8 | 3.4 | 4.4 | 4.5 | 4.5 | 4.0 |
ug total | 15.0 | 17.3 | ||||||
DpnII Digestion
- Reaction Table
V6S2A | V6S2B | V6S2C | V6S2D | V6S3A | V6S3B | V6S3C | V6S3D | |
ng/uL | 99.9 | 95.4 | 94.6 | 84.5 | 110 | 112.2 | 111.6 | 99.8 |
ug in 35 uL | 3.5 | 3.3 | 3.3 | 3.0 | 3.9 | 3.9 | 3.9 | 3.5 |
uL sample | 35 | 35 | 35 | 35 | 35 | 35 | 35 | 35 |
uL 10X buffer | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
uL nfH20 | 8 | 8 | 8 | 8 | 8 | 8 | 8 | 8 |
uL DpnII (10 U/uL) | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 |
Total | 50 | 50 | 50 | 50 | 50 | 50 | 50 | 50 |
- Incubate 16 hours at 37C
- Heat inactivate 10 minutes at 65C