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.