Bisulflash Kit Test[edit]
- Athurva and I will test the bisulflash kit from Epigentek
- We will perform bisulfite conversion on 100, 10 & 1 ng of Jurkat gDNA with NTC
- We will verify the completeness of bisulfite conversion based on quantitative qPCR and MDA
Bisulfite Conversion[edit]
- Done by Athurva
- Elute each samples with 15ul of elution buffer and we will use 5ul for qPCR, 5ul for MDA and 2 ul for a gel quantification
qPCR Analysis with Primer set CHR22[edit]
- We picked CHR 22 set, since it has the smallest amplicon size among the 3 sets of primers we have, bisulfite conversion by bisulFlash kit produces ~200bp fragments, chances of primer sites being separated is very possible.
Content
|
Volume per Reaction
|
MM
|
qPCR Setup
|
2X iQ Supermix |
20 |
88 |
NTC
|
Primer 22F |
6 |
26.4 |
1ng
|
Primer 22R |
6 |
26.4 |
10ng
|
gDNA |
5 |
Do not add to MM |
100ng
|
H2O |
3 |
13.2 |
|
Total |
40 |
154 |
|
- Add 35ul MM to each samples
Step1 96C, 3m
Step2 95C, 30s
Step3 62C, 1m
Step4 72C, 1m
Step5 Go to step2 repeat 39 times
Step6 72C, 5m
Step7 15C, Forever
MDA Analysis[edit]
- Denature all samples at 95C for 30sec and quickly transfer to cooler rack
Content
|
Volume per Reaction
|
MM
|
10X Repli Phi Buffer |
2 |
8.8
|
1mM N9 Primer |
1 |
4.4
|
25mM dNTP |
0.8 |
3.52
|
Repliphi phi 29 (100U/ul) |
1 |
4.4
|
Klenow Exo Minus (10U/ul) |
0.5 |
2.2
|
50X SYBG |
0.1 |
0.44
|
H2O |
9.6 |
42.24
|
Modified DNA Sample |
5 |
Do not add to MM
|
Total |
20 |
66
|
- Add 15ul of MDA mastermix to each sample
- 30C 120 min, 65C 10 min, 15C hold
PAGE Gel Analysis[edit]
File:ZhangLab 2 2011-09-08 10hr 40min.jpg
Results[edit]
The bisulfite conversion is successful, verfied by the PCR validation test. Yet sample loss is a huge issue for this kit, we dont obtain expected yield from the bisuflash kit.