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

From ZhangLabWiki
Jump to navigation Jump to search
>Djacobse
>Djacobse
 
Line 208: Line 208:
|}
|}


  2. Incubate samples at 37C for 16 hours; 20 minute heat shock at 65C; Continued [[Daniel:Notebook/HiResChrPaint/2013-9-4|Continued 09/04/2013]]
  2. Incubate samples at 37C for 16 hours; 20 minute heat shock at 65C; Continued [[Daniel:Notebook/HiResChrPaint/2013-9-4|09/04/2013]]

Latest revision as of 00:04, 4 October 2013

BSPP Control[edit]

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.

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]

File:2013-09-03-BSPP.png

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