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.