Kun:LabNotes/ASE/2008-1-6

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Exp.1 Real-time PCR on the capturing reactions from 01/02/08 and 01/03/08's reactions[edit]

                         x 20           x 2
    Template:            10ul     or    15ul
    10X buffer:           9ul     or    14ul
    25mM dNTP:          0.8ul     or   1.2ul
    100uM AmpV6.2SolF   0.4ul     or   0.6ul
    100uM AmpV6.2SolR   0.4ul     or   0.6ul
    50X SYBGI:          0.4ul     or   0.6ul
    JumpStart Taq         2ul     or     3ul
    H2O:                 77ul     or   115ul

94C 3min -> (94C 45 sec -> 57C 1min -> 72C 1min) x 10 -> (94C 30min -> 72C 1min) x 8 -> 72C 3min -> 15C hold.

The amplification curves didn't come up at the end of PCR. Then I realized that I should be using the AmpSolF2/AmpSolR primer pair. I added 0.5ul of 100uM primers to each reaction, repeat the PCR. The amplification curves came up, but I saw many non-specific bands in the PAGE gel.

File:2008-01-07-eSNP84k-capture-PCR.png