Kun:LabNotes/ASE/2007-12-3

From ZhangLabWiki
Jump to navigation Jump to search

Exp.1 Resubmit PCR amplicons for sequencing[edit]

I'm resubmitting three amplicons that were failed to sequence in the 11/30/2007 sample submission.

  1. rs1045216 GC1F, I used the reverse PCR primer as the sequencing primer;
  2. rs2271504 GC1F, I used the forward PCR primer as the sequencing primer;
  3. rs2271504 GC1EP, I used the forward PCR primer as the sequencing primer;

Exp. 2 Repeat the circularization experiment using the CES22k probe set on the cDNAs from Hues6, H1 hybrid, H2 hybrid.[edit]

I decided to repeat this experiment because there is still a noticeable level of stochasticity in allelic ratios based on the recent Solexa run on GM20431 cDNAs. I think the drift of allelic ratios will be reduced by combine the captured DNA from two independent reactions. I'm doing this on cDNAs only because I can still make genotyping calls with a reasonable accuracy with the current level of circularization efficiency.


Template 10x buffer CES22k probe #4(358nM) H2O
1 Hues6 cDNA(155ng/ul) 0.3ul 1ul 4.2ul 4.0ul
2 H1 cDNA(35ng/ul) 1.4ul 1ul 4.2ul 2.9ul
3 H2 cDNA(48ng/ul) 1ul 1ul 4.2ul 3.3ul

95C 10min->60C 18h-> add 0.5ul 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 1ul of exo mix (Exo I, Exo III, T7 exo)->37c 1 hour -> 94C 5min -> 4C hold.