Kun:LabNotes/ASE/2008-6-25: Difference between revisions
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m (New page: ==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 [[Kun:LabNotes/ASE/2007-1...) |
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**Titatium Taq: 2U/ul | **Titatium Taq: 2U/ul | ||
**AmpLigase: 0.5U/ul | **AmpLigase: 0.5U/ul | ||
**dNTP: 1uM | **dNTP: 1uM (each) | ||
{| border="1" cellpadding="5" cellspacing="0" align="center" | |||
|- | |||
| align="center" style="background:#f0f0f0;"|''' ''' | |||
| align="center" style="background:#f0f0f0;"|''' ''' | |||
| align="center" style="background:#f0f0f0;"|'''Template''' | |||
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. | | align="center" style="background:#f0f0f0;"|'''10x buffer''' | ||
| align="center" style="background:#f0f0f0;"|'''CES22k''' | |||
| align="center" style="background:#f0f0f0;"|'''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=== |
Revision as of 17:18, 25 June 2008
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.