Kun:LabNotes/ASE/2008-6-25
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Repeat CES22k SNP capture[edit]
- 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.
- I just realized that the 10mM dNTP from ABI is for total concentration of all four nucleotides. So it is actually 2.5mM for each. Most experiment did in the past eight months were based on the assumption that it's 10mM each. That's probably why the yield of PCR I got with Jumpstart Taq or Platinum Taq was lower than with Taq master mix. Also the dNTP concentration in KLN/SLN mix was 1/4 of the concentration in my calculation. I decided to increase the dNTP concentration by 4-fold to 1uM in today's experiment.
Circularization[edit]
- 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 | Hues56 gDNA (37ng/ul)x5 | 6ul | 1ul | 1.2ul | 1.8ul |
E | Hues59 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[edit]
PCR x 30 Template: 12ul 2x iProof master mix: 50ul 1500 100uM AmpF2Sol: 0.4ul 12 100uM AmpR2Sol: 0.4ul 12 50X SYBG I: 0.4ul 12 H2O 37ul 1110
98C 30sec -> (98C 10sec -> 58C 20sec -> 72C 10sec) x 8 -> (98C 10sec -> 72C 20sec) x 9 -> 72C 3min -> 4C hold.
PAGE purification of 80ul unpurified A1&A2, cut out both bands for monomer and dimers. Quantification of both bands. monomer dimer A1: 4.9ng/ul 4.2ng/ul PGP1Fa1 (77nM) A2: 11ng/ul 9.6ng/ul PGP1Fb1 (174nM)
Purified 200ul of A1, A2, B, C, D, E with DNA concentrator-25 columns. Yield: A1: 38ng/ul x 30ul ==> clonewell purification of 20ul; 8.6ng/ul (89nM) PGP1Fa2 ==> sequenced A2: 44ng/ul x 30ul ==> clonewell purification of 20ul; 6.4ng/ul (66nM) PGP1Fb2 ==> sequenced B: 20ng/ul (125nM) x 30ul ==> submitted directly for sequencing C: 39ng/ul (274nM) x 30ul ==> submitted directly for sequencing D: 50ng/ul (312nM) x 30ul ==> submitted directly for sequencing E: 43ng/ul (268nM) x 30ul ==> submitted directly for sequencing
Note: to calculate the concentration for the non-size-selected amplicons, I used the MW of the smallest band, then multiply the concentration with 0.6 (just a rough estimate).