Daniel:Notebook/HiResChrPaint/2013-9-3
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BSPP Control[edit]
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.
Samples
- BSPP 1-DMR 220K Exp 1 (10nM); 2/8/11 from Noi
- BSPP 2-DMR 220K Exp 2 (10nM); 2/8/11 from Noi
qPCR Amplification[edit]
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[edit]
Nanodrop Results[edit]
Probe | BSPP 1A | BSPP 1B | BSPP 1C | BSPP 1D | BSPP 2A | BSPP 2B | BSPP 2C | BSPP 2D |
ng/uL | 68.8 | 75.8 | 70.1 | 71.8 | 61.4 | 61.8 | 60.3 | 62.7 |
ug in 40 uL | 2.8 | 3.0 | 2.8 | 2.9 | 2.5 | 2.5 | 2.4 | 2.5 |
Total | 11.5 | 9.8 |
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 | 68.8 | 75.8 | 70.1 | 71.8 | 61.4 | 61.8 | 60.3 | 62.7 |
ug in 35 uL | 2.4 | 2.7 | 2.5 | 2.5 | 2.1 | 2.2 | 2.1 | 2.2 |
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 09/04/2013