Kun:LabNotes/CpgSeq/2007-11-12

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Exp. 1 Phosphorylation of CpG MIP probes[edit]

   Set I (128nM):         13ul
   Set II (162nM):         5ul
   10X T4 Ligase Buffer: 2.5ul
   PNK:                    2ul
   H2O:                  2.5ul

37C 15min -> 75C 10min -> make 1:10 dilution to 10nM

Exp. 2 CpG island capture[edit]

               RL DNA         Jurkat DNA (all converted, conc. unknown)
    Template    15ul            15ul
    10x buffer   2ul             2ul
    10nM CpG-MIP 1ul             1ul

94C 3min->60C 12h-> add 2.5ul/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 3ul of exo mix (Exo I, Exo III, T7 exo)->37c 1 hour -> 94C 5min -> 4C hold.