Kun:LabNotes/CpgSeq/2007-11-11
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Exp.l PCR on the captured CpG islands[edit]
- Template: two capturing reactions started at 11/09/2007.
- Primer: Amp_F_sol, Amp_R_sol
x 1 x 2 template: 5ul 10ul ABI 10x buffer: 4.5ul 9ul 25mM MgCl2: 3ul 6ul 10mM dNTP: 1ul 2ul 100uM AmpFSol: 0.15ul 0.3ul 100uM AmpRSol: 0.15ul 0.3ul 50X SYBG I: 0.4ul 0.8ul iTaq: 1ul 2ul H2O: 35.3ul 70.6ul
94C 3min -> 10 cycles of (94C 45sec -> 59C 2min -> 72C 1min) -> 12 cycles of (94C 45 sec -> 72C 1min) -> 72C 3min -> 15C hold.
No amplification. I realized that I didn't phosphorylate the 5'-end of the probes. So no circularization is not a surprise.
Exp. 2 Repeat the bisulfite conversion of genomic DNA with EpiTect kit[edit]
Genomic DNA samples (conc. measured by Nanodrop):
- DNA RL (from Dr. Huidong Shi), 185ng/ul
- Jurkat gDNA (from NEB), 194ng/ul
Resuspend one vial of Bisulfite Mix in 800ul nuclease free water, votex for 5min. Set up reactions:
RL Jurkat DNA 5ul x 2 5ul x 2 Water 15ul x 2 15ul x 2 Bisulfite mix 85ul x 2 85ul x 2 DNA Protection Buffer 35ul x 2 35ul x 2
Perform the bisulfite conversion on PTC-200 using the EpiTect program 99C5min->60C20min->99C5min->60C1h25min->99C5min->60C2h55min->20Chold.
Follow the EpiTect protocol to clean up the bisulphite converted DNAs.