Kun:LabNotes/ASE/2008-6-25

From ZhangLabWiki
Jump to navigation Jump to search

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).