Kun:LabNotes/ASE/2008-6-25

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Repeat CES22k SNP capture

I decided to repeat the circularization reaction with a slightly different protocol. The reason is that I found the results from the PGP1L-011507 experiment was quite good. My hypothesis is that if the poor efficiency is due to the probes anneal to non-specific targets and get extended from the 3'-end, then adding KLN mix later will give more time for the probes to find their targets.

Circularization

  • dscDNA: 100ng per reaction, gDNA:Probe=1:200.
  • dNTP: 2x theoretical number of circles = 1pmole/10ul = 100nM.
  • Probe: CES22k, 06/05/08, 525nM.
  • KLN mix:
    • Titatium Taq: 2U/ul
    • AmpLigase: 0.5U/ul
    • dNTP: 1uM (each)
Template 10x buffer CES22k H2O
A PGP1F cDNA(dT/RNA primed,102ng/ul, 06/24/08)x10 1ul 1ul 1.2ul 6.8ul
B Hues56 cDNA(dT/RNA primed,70ng/ul, 06/18/08)x5 1.4ul 1ul 1.2ul 6.4ul
C Hues59 cDNA(dT/RNA primed,49ng/ul, 06/18/08)x5 1.6ul 1ul 1.2ul 6.2ul
D Hues58 gDNA (37ng/ul)x5 6ul 1ul 1.2ul 1.8ul
E Hues66 gDNA (30ng/ul)x5 7ul 1ul 1.2ul 0.8ul

94C 5min -> 60C 24h -> add 1ul KLN mix -> 60C 1h -> 20 cycles of (94C 1min -> 0.1C/sec to 60C -> 60C 1h) -> 60C hold -> 94C 1min -> 37C 1min -> add 1ul Exo I&III -> 37C 2h -> 94C 5min -> 4C hold.

PCR

  PCR                             x 10
  Template:               12ul
  2x iProof master mix:   50ul     500
  100uM AmpF2Sol:        0.4ul       4
  100uM AmpR2Sol:        0.4ul       4
  50X SYBG I:            0.4ul       4
  H2O                     37ul     370

98C 30sec -> (98C 10sec -> 58C 20sec -> 72C 10sec) x 8 -> (98C 10sec -> 72C 20sec) x 9 -> 72C 3min -> 4C hold.