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
x10 PGP1F-dsCDNA (06/24/08 A, 102ng/ul): 1ul 10 CES22k probe (525nM): 1.2ul 12 10X buffer: 1ul 10 H2O: 6.8ul 68
94C 5min -> 60C 24h -> 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.