Kun:LabNotes/ASE/2007-12-3
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.
- rs1045216 GC1F, I used the reverse PCR primer as the sequencing primer;
- rs2271504 GC1F, I used the forward PCR primer as the sequencing primer;
- 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.