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.