Athurva Gore/LabNotes/2011-9-8
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Low-input Bisulfite Conversion[edit]
- Repeated experiments from last week had an issue
- DNA was totally unconverted, in all samples
- Alan's separate experiments had the same issue
- Problem with bisulfite preparation?
- Alan mixed tube, probably not a problem though since mixing is very simple
- Reagents too old? This would imply Imprint has an extremely short shelf life
- Ordered more from Sigma; will hopefully receive soon
Epigentek Bisulflash Kit[edit]
- Earlier results (from 7/14/2011), comparing Epigentek to Zymo
File:Zymo vs Epigentek Edited 07142011.png
- Epigentek kit did not work well
- Decided to try Bisulflash kit again with Alan
- Hoped it would work better this time
Protocol[edit]
- Bisulfite converted 100 ng, 10 ng, and 1 ng of genomic DNA (Jurkat); also converted water (NTC)
- Followed epigentek protocol (95 C for 20 minutes incubation in sodium bisulfite, column purification with desulphonation)
- Performed two experiments:
qPCR Experiment[edit]
- From Alan's Notebook:
- 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 Experiment[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
Gel Image[edit]
File:Epigentek Normal MDA PCR.png
MDA Results[edit]
- Only 100 ng sample worked well
- 10 ng sample has a small visible presence after 2 hours of MDA
- Library size seems to be spread out - not centered around 200-ish like sales rep said earlier...
- Very few molecules appear to be left in 1 ng sample...does not bode well for Epigentek kit
PCR Results[edit]
- For some reason, 10 ng sample did not amplify as well as 1 ng sample
- Could have been switched, or could be some issues with PCR setup
- However, some DNA was observed. Not as much as with Zymo kit though; loss is either greater or library size is smaller (though MDA does not agree with this)