Kun:LabNotes/ASE/2008-7-27: Difference between revisions

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m (New page: ==Perform additional CES22k capturing reactions on GM10835 and PGP1L cDNAs== ===SNP capture=== GM10835(mRNA) GM10835(fr...)
 
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===SNP capture===
===SNP capture===
                                    
                                    
                                GM10835(mRNA) GM10835(frag mRNA)  PGP1L(mRNA)      PGP1L(total RNA)
                                GM10835(mRNA) GM10835(frag mRNA)  PGP1L(mRNA)      PGP1L(total RNA)
                                    x4                 x4             x4                 x4
                                36ng/ul x4       7.2ng/ul x4     16ng/ul x4         68ng/ul x4
   ds-cDNA                        1.5ul              1.5ul          1.5ul              1.5ul
   ds-cDNA                        1.5ul              1.5ul          1.5ul              1.5ul
   10X Ampligase Buffer            1.0ul              1.0ul          1.0ul              0.4ul
   10X Ampligase Buffer            1.0ul              1.0ul          1.0ul              0.4ul

Revision as of 22:32, 27 July 2008

Perform additional CES22k capturing reactions on GM10835 and PGP1L cDNAs

SNP capture

                               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

  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.