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.