Daniel:Notebook/HiResChrPaint/2013-10-22
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BSPP Control[edit]
- For first production of BSPP control, see 09/03/2013
Since I'm out of control group, I'm going to make some more.
qPCR[edit]
1. Reaction Table
10 nM OligoPool | 2X Kapa SYBR qPCR MM | 100 uM AP1V61U-Amino | 100 uM AP2V6 | H20 | Total Volume | |
per rxn | 0.4 | 50 | 0.4 | 0.4 | 48.8 | 100 |
BSPP 1 (12.5x) | 5 | 625 | 5 | 5 | 610 | 1250 |
BSPP 2 (12.5x) | 5 | 625 | 5 | 5 | 610 | 1250 |
2. Aliquot 100 uL into each of 12 qPCR tubes 3. PCR protocol: i. PCR 3 min 95C ii. 95C 3 sec iii. 55C 30 sec iv. 72C 20 sec v. plate read vi. goto ii x17 vii. 72C 2 min viii. 16C hold 4. Pooled 3 samples together each (300 uL each, 4 pools per set, 8 total) 5. Column Purification, elute with 40 uL EB 6. Measure concentrations in nanodrop
qPCR Results[edit]
File:2013-10-22-BSPPControl.png
Nanodrop Results[edit]
Probe | BSPP 1A | BSPP 1B | BSPP 1C | BSPP 1D | BSPP 2A | BSPP 2B | BSPP 2C | BSPP 2D |
ng/uL | 97.9 | 94.5 | 96.9 | 91.9 | 90.2 | 92.5 | 90.7 | 82.4 |
ug in 40 uL | 3.9 | 3.8 | 3.9 | 3.7 | 3.6 | 3.7 | 3.6 | 3.3 |
Total | 15.2 | 14.2 |
DpnII Digestion[edit]
1. Reaction Table
Sample | BSPP 1A | BSPP 1B | BSPP 1C | BSPP 1D | BSPP 2A | BSPP 2B | BSPP 2C | BSPP 2D |
ng/uL | 97.9 | 94.5 | 96.9 | 91.9 | 90.2 | 92.5 | 90.7 | 82.4 |
ug in 35 uL | 3.4 | 3.3 | 3.4 | 3.2 | 3.2 | 3.2 | 3.2 | 2.9 |
uL 10X buffer | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
uL DpnII (50U/uL) | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 |
uL nfH2O | 8 | 8 | 8 | 8 | 8 | 8 | 8 | 8 |
Total | 50 | 50 | 50 | 50 | 50 | 50 | 50 | 50 |
2. Incubate samples at 37C for 16 hours; 20 minute heat shock at 65C; Continued tomorrow