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.