Kun:LabNotes/ASE/2008-7-27

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Perform additional CES22k capturing reactions on GM10835 and PGP1L cDNAs[edit]

SNP capture[edit]

                               GM10835(mRNA)  GM10835(frag mRNA)  PGP1L(mRNA)      PGP1L(total RNA)
                                36ng/ul x4        7.2ng/ul x4     16ng/ul x4          68ng/ul x4
  ds-cDNA                         1.5ul              1.5ul           1.5ul              1.5ul
  10X Ampligase Buffer            1.0ul              1.0ul           1.0ul              0.4ul
  CES22k probe(325nM,AL,07/13/08) 1.2ul              1.2ul           1.2ul              1.2ul
  H2O                             6.3ul              6.3ul           6.3ul              6.9ul
  95C 5min -> 0.1C/sec to 60C -> 60C 20h -> 55c 1min -> add 1ul SLN mix (N=1uM) 
  -> 55c 30min -> 94C 1min -> 37C 1min -> add 1ul Exo I&III -> 37C 2h -> 94C 5min.

PCR[edit]

  PCR                      x 4 x 4
  Template:               12ul
  2x iProof master mix:   50ul    
  100uM AmpF2SolH:       0.4ul    
  100uM AmpR2SolH:       0.4ul    
  50X SYBG I:            0.4ul    
  H2O                     37ul    

98C 30sec -> (98C 10sec -> 58C 20sec -> 72C 10sec) x 8 -> (98C 10sec -> 72C 20sec) x 11 -> 72C 3min -> 4C hold.

  File:2008-07-28-CES22k-capture-PCR.png
  Pooled 5x50ul for each sample (incl. amplicons from 07/26/08), purified with DNA concentrator-25.
  Yield:           assuming 60% are monomer, 30% dimer, 10% non-specific
  A: GM10835m:   32ng/ul x 40ul   272nM
  B: GM10835mf:  12ng/ul x 40ul   102nM
  C: PGP1Fm:     31ng/ul x 40ul   264nM
  D: PGP1Fdt:    21ng/ul x 40ul   179nM