Daniel:Notebook/HiResChrPaint/2013-9-3
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BSPP Control
At Dr. Zhang's recommendation I'm creating negative controls using Dinh/Noi's bisulfite padlock probes (BSPPs). I have obtained the probes from Noi and I have the primers, so I'm going to proceed making probes as I usually do. These probes, however, should not anneal to regular human DNA, as they have been modified for methylation detection. I'm going to be splicing in aminoallyl dUTP and dye-coupling the probes, as usual. I'll also be performing DpnII Digestion and Lambda Exonuclease digestion, as I usually do.
qPCR Amplification
1. Reaction Table
10 nM OligoPool | 2X Kapa SYBR qPCR MM | 100 uM AP1V41U | 100 uM AP2V4 | 100 uM AP1V61U | 100 uM AP2V6 | 2 mM aa-dUTP | H20 | Total Volume | |
per rxn | 0.2 | 50 | 0.4 | 0.4 | 0.4 | 0.4 | 2.5 | 45.7 | 100 |
BSPP 1 (12.5x) | 2.5 | 625 | 5 | 5 | 0 | 0 | 31 | 581.5 | 1250 |
BSPP 2 (12.5x) | 2.5 | 625 | 0 | 0 | 5 | 5 | 31 | 581.5 | 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