Daniel:Notebook/HiResChrPaint/2013-9-3: Difference between revisions

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[[Image:2013-09-03-BSPP.png|600px]]
[[Image:2013-09-03-BSPP.png|600px]]
===Nanodrop Results===
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="font-size:12pt;font-weight:bold" align="center"
| width="65" height="15" | Probe
| width="65" | BSPP 1A
| width="65" | BSPP 1B
| width="65" | BSPP 1C
| width="65" | BSPP 1D
| width="65" | BSPP 2A
| width="65" | BSPP 2B
| width="65" | BSPP 2C
| width="65" | BSPP 2D
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | ng/uL
| align="center" | 68.8
| align="center" | 75.8
| align="center" | 70.1
| align="center" | 71.8
| align="center" | 61.4
| align="center" | 61.8
| align="center" | 60.3
| align="center" | 62.7
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | ug in 40 uL
| align="center" | 2.8
| align="center" | 3.0
| align="center" | 2.8
| align="center" | 2.9
| align="center" | 2.5
| align="center" | 2.5
| align="center" | 2.4
| align="center" | 2.5
|- style="font-size:12pt" align="center"
|style="font-weight:bold" height="15" | Total
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | 11.5
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | 9.8
|}
==DpnII Digestion==

Revision as of 23:39, 3 September 2013

BSPP Control

Back to Calendar

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

Nanodrop Results

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