Kun:LabNotes/CpgSeq/2007-12-20
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Exp.1 PCR on bisulfite converted DNA[edit]
Ten PCR primer pairs used in Huidong's Cancer Res. paper arrived. Set up PCR on converted and non-converted DNA. Templates:
- Converted RL, 1:100 dilution, 12/05/07
- Converted Jurkat, 1:100 dilution, 12/05/07
- Non-converted Jurkat DNA, 1ng/ul
One 30 Template: 1ul 10X Jumpstart buffer: 2.5ul 75ul 10uM primer mix: 1ul 10x3ul 10mM dNTP: 0.5ul 15ul JumpStart Taq: 0.25ul 7.5ul H2O: 19.75ul 600ul
94C 3min -> 8 cycles of (94C 30sec -> 64C 30sec (-1C per cycle) -> 72C 30 sec) -> 27 cycles of (94C 30sec -> 56C 30sec -> 72C 30 sec) -> 72C 3min -> 15C hold.
File:2007-12-20-Bis-PCR.png
I added 8 more PCR cycles, then pooled 15ul of the eight Jurkat bis amplicons, purified with Bioneer's column. Nanodrop read: 19ng/ul x 30ul.