Kun:LabNotes/CpgSeq/2007-12-23
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Exp.1 PCR on the CpGMIP capturing reaction.[edit]
one reaction 4 reactions Template: 5ul each 10x Buffer: 4.5ul 18ul 25mM dNTP 0.6ul 2.4ul 100uM AmpSolF 0.2ul 0.8ul 100uM AmpSolR 0.2ul 0.8ul JumpStart Taq 0.5ul 4ul 50X SYBG I 0.2ul 0.8ul H2O 38.8ul 156ul
94C 3min -> 8 cycles of (94C 45sec -> 58C 1min -> 72C 1min) -> 10 cycles of(94C 30min -> 72C 1min)-> 72C 3min -> 15C hold.
File:2007-12-24-CpGMIPv2-capture-PCR.png
I think the MIP probes did capture the artificial targets even in the presence of bisulfite converted genomic DNA. The reason I didn't see anything in D is probably due to the fact that I have too few probes.
I decided to do PAGE purification on #3 and #4, then do another round of PCR.
File:2007-12-27-CpGMIPv2-capture-PCR-PAGE.png
The gel slices were sheared and then soaked with 200ul 1X PCR buffer at 4C overnight.