BSPP Control[edit]
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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.
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