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.