Kun:LabNotes/CpgSeq/2008-1-10
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Exp.1 Random-primed synthesis of bis-DNA[edit]
Since I failed to get anything in the last CpGMIP capturing experiment, I decided to do two rounds of N6-primed synthesis of the bisulfite converted DNA, and repeat the capturing reactions.
Bis-JK(01/02/08) Bis-RL(01/02/08) DNA 8ul 8ul 10X Klenow buffer 4ul 4ul 10mM dNTP 4ul 4ul H2O 18ul 18ul 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: 200ng/ul x 20ul Bis-RL: 122ng/ul x 20ul
Exp.2 CpgMIP capture[edit]
RL DNA(122ng/ul) Jurkat DNA(200ng/ul) RL-PCR8(0.1nM) Template 7ul 7ul 1ul + 6ul H2O 10x buffer 1ul 1ul 1ul 10nM CpG-MIP 2ul 2ul 2ul
RL DNA(122ng/ul) Jurkat DNA(200ng/ul) RL-PCR8(0.1nM) Template 7ul 7ul 1ul + 6ul H2O 10x buffer 1ul 1ul 1ul 10nM CpG-MIPv2 2ul 2ul 2ul
95C 10min->55C 20h-> add 1ul SLN mix (1U/ul stoffel, 0.5U/ul AmpLigase, 200nM dNTP) -> 55C 30min -> 15 cycles of (94C 1min -> 55C 1h) -> 55C 4h -> 37C 1min -> add 1ul of exo mix (Exo I, Exo III, T7 exo)->37c 1 hour -> 94C 5min -> 4C hold.