Kun:LabNotes/CpgSeq/2007-11-9

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Exp. 1 Perform PCR on the bisulfite converted DNAs (positive control)[edit]

  • Template: Jurkat DNA, DNA RL, concentration unknown.
  • Primer: hd48MIP-1, hd48MIP-2, hd48MIP-5.
                            x1         x6
        template:           1ul       each
        10x ABI buffer:   2.5ul       15ul
        25mM MgCl2:       1.5ul        9ul
        10mM dNTP:        0.5ul        3ul
        10uM Primer mix:  0.5ul      1ulx3 
        50X SYBG I:       0.2ul      1.2ul
        iTaq:             0.2ul      1.2ul
        H2O:             18.6ul      114ul

94C 3min -> 40 cycles of (94C 30sec -> 57C 30min -> 72C 1min) -> 72C 3min -> 15C hold.

Exp. 2 Measure the concentration of PAGE purified padlock probes by gel electrophoresis[edit]

   Set 1: 3.7ng/ul = 128nM
   Set 2: 4.2ng/ul = 162nM

Mix two sets in 2:1 molar ratio, and dilute to 1nM (total concentration):

   13ul of set I + 5ul of Set 2 + 230ul H2O -> 10nM CpG-MIP mix
   20ul of 10uM mix + 180ul H2O -> 1nM CpG-MIP mix

Exp. 3 CpG island capture[edit]

               RL DNA         Jurkat DNA (all converted, conc. unknown)
    Template    15ul            15ul
    10x buffer   2ul             2ul
    1nM 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.