Kun:LabNotes/ASE/2007-12-3: Difference between revisions
(New page: ==Exp.1 Resubmit PCR amplicons for sequencing== I'm resubmitting three amplicons that were failed to sequence in the 11/30/2007 sample submission. # rs1045216 GC1F, I used the reverse PCR...) |
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| align="center" style="background:#f0f0f0;"|'''H2O''' | | align="center" style="background:#f0f0f0;"|'''H2O''' | ||
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|1||Hues6 cDNA(155ng/ul)||0.3ul||1ul||4.2ul|| | |1||Hues6 cDNA(155ng/ul)||0.3ul||1ul||4.2ul||4.0ul | ||
|- | |- | ||
|2||H1 cDNA(35ng/ul)||1.4ul||1ul||4.2ul||2. | |2||H1 cDNA(35ng/ul)||1.4ul||1ul||4.2ul||2.9ul | ||
|- | |- | ||
|3||H2 cDNA(48ng/ul)||1ul||1ul||4.2ul|| | |3||H2 cDNA(48ng/ul)||1ul||1ul||4.2ul||3.3ul | ||
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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. | 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. |
Latest revision as of 22:01, 3 December 2007
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.