Daniel:Notebook/HiResChrPaint/2013-9-3

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Revision as of 22:21, 3 September 2013 by >Djacobse (→‎qPCR Results)
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BSPP Control

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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 AP1V61U 100 uM AP2V6 2 mM aa-dUTP H20 Total Volume
per rxn 0.4 50 0.4 0.4 2.5 46.3 100
BSPP 1 (12.5x) 5 625 5 5 31 579 1250
BSPP 2 (12.5x) 5 625 5 5 31 579 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

File:2013-09-03-BSPP.png