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