Kun:LabNotes/CpgSeq/2007-12-5
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Exp.1 Repeat the PAGE purification of CpGMIPv2 oligos[edit]
I lost the CpGMIPv2L oligos in yesterday's purification. So I repeated the PAGE purification experiment. The pelletes were resuspended in 20ul ddH2O. I use 3ul to for PAGE quantification. I added 2ul 10X T4 ligase buffer and 1ul T4PNK to the remaining 17ul. Incubate at 37C 15min, 85C 20min.
CpGMIPv2 S1(95nM): 17ul CpGMIPv2 S2(211nM) 20ul -> 6.3ul CpGMIPv2 L1(243nM) 20ul -> 2.7ul H2O 191ul ---------- 200ul x 10nM
Exp. 2 CpG island capture[edit]
RL DNA(103ng/ul) Jurkat DNA(94ng/ul) Template 10ul 10ul 10x buffer 1.5ul 1.5ul 10nM CpG-MIP 2ul 2ul H2O 1.5ul 1.5ul
94C 3min->60C 20h-> add 1ul/1.5ul SLN mix (1U/ul stoffel, 0.5U/ul AmpLigase, 100nM dNTP) -> 60C 4h -> 15 cycles of (94C 1min -> 60C 1h) -> 60C 4h -> 37C 1min -> add 1ul of exo mix (Exo I, Exo III, T7 exo)->37c 1 hour -> 94C 5min -> 4C hold.