Kun:LabNotes/CpgSeq/2007-12-10
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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