Kun:LabNotes/CpgSeq/2008-1-26
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Exp.1 Random-primed synthesis of bis-DNA[edit]
Bis-JK(01/25/08) Bis-RL(01/25/08) DNA 20ul 20ul 10X Klenow buffer 4ul 4ul 10mM dNTP 4ul 4ul H2O 8ul 8ul 1mM N6 primer 2ul 2ul 95C 2min -> 16C 1min -> add 2ul 20U/ul Klenow exo- -> 16C 15min -> 37C 10min -> 95C 2min -> 16C 1min -> add 2ul 20U/ul Klenow exo- -> 16C 15min -> 37C 10min Purify with MinElute column. Yield: Bis-JK: 145ng/ul x 14ul Bis-RL: 209ng/ul x 14ul
Exp.2 Cpg capture[edit]
RL dsDNA(209ng/ul) Jurkat dsDNA(145ng/ul) RL ssDNA Jurkat ssDNA Template 6ul 6ul 12ul 12ul 10x buffer 2ul 2ul 2ul 2ul 98nM CpG28k 4ul 4ul 4ul 4ul H2O 6ul 6ul 0ul 0ul
95C 10min -> 60C 40h -> add 2.5ul NSL mix (Stoffel 2U/ul, AmpLigase 0.5U/ul, dNTP 50uM) -> 60C 1h - -> 37C 1min -> add 1ul Exo I&&III, 1ul T7 exo, 37C 2h -> 94C 5min -> 4C hold.