Kun:LabNotes/CpgSeq/2007-12-20

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Exp.1 PCR on bisulfite converted DNA[edit]

Ten PCR primer pairs used in Huidong's Cancer Res. paper arrived. Set up PCR on converted and non-converted DNA. Templates:

  1. Converted RL, 1:100 dilution, 12/05/07
  2. Converted Jurkat, 1:100 dilution, 12/05/07
  3. Non-converted Jurkat DNA, 1ng/ul
                               One      30
      Template:                1ul
      10X Jumpstart buffer:  2.5ul      75ul
      10uM primer mix:         1ul    10x3ul
      10mM dNTP:             0.5ul      15ul 
      JumpStart Taq:        0.25ul     7.5ul
      H2O:                 19.75ul     600ul

94C 3min -> 8 cycles of (94C 30sec -> 64C 30sec (-1C per cycle) -> 72C 30 sec) -> 27 cycles of (94C 30sec -> 56C 30sec -> 72C 30 sec) -> 72C 3min -> 15C hold.

 File:2007-12-20-Bis-PCR.png

I added 8 more PCR cycles, then pooled 15ul of the eight Jurkat bis amplicons, purified with Bioneer's column. Nanodrop read: 19ng/ul x 30ul.