Kun:LabNotes/CpgSeq/2007-12-10

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Revision as of 20:39, 10 December 2007 by KunZhang (talk | contribs) (New page: ==Exp. 1 PAGE purification on the CpG MIP PCR amplicons did on 12/09/2007== I hope that one of the 300- or 400+ bands are from the captured CpG sequences. To confirm this, I will do PAGE p...)
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Exp. 1 PAGE purification on the CpG MIP PCR amplicons did on 12/09/2007

I hope that one of the 300- or 400+ bands are from the captured CpG sequences. To confirm this, I will do PAGE purification, then re-amplify, PCR cloning and sequence a few clones.

Exp. 2 Confirmation of CpG MIP capturing experiment by PCR

Another way to confirm the result of the MIP capturing experiment is to perform PCR on the circularization reactions with target-specific PCR primers. I will use the bisulfite converted DNA as positive controls. The primers used included HD48MIP 1-3.

   Template:            1ul  (1:100 dilution of bifulfite converted gDNAs)
   10X buffer:        2.5ul
   10mM dNTP:         0.5ul
   10uM Primer pair   0.5ul
   50X SYBR I         0.2ul
   JumpStart Taq     0.25ul
   H2O                 20ul

Total number of reactions: 4 MIP reactions x 3 primer pairs + 2 control x 3 primer pairs = 18

94C 3min -> 30 cycles of (94C 30min -> 57C 45sec -> 72C 30sec) -> 72C 3min