Kun:LabNotes/ASE/2007-12-9
Exp. 1 Perform CES22k eSNP capture on PGP and hybrid samples[edit]
Genomic targets: 800ng/3pg per haploid genome = 2.67x10^5 copies = 2.22x10~-19 moles Probes: 2.22x10~-19 moles * 22,000 * 125 = 1.22x10^-12 moles.
Template | 10x buffer | CES22k probes #5 (127nM) | H2O | ||
1 | GM20431 gDNA(347ng/ul) | 2.3ul | 1.5ul | 9ul | 0.7ul |
2 | GM20431 cDNA (121ng/ul) | 0.8ul | 1.5ul | 9ul | 2.2ul |
3 | GC1F cDNA(48ng/ul) | 2ul | 1.5ul | 9ul | 1ul |
4 | GC1EP cDNA(52ng/ul) | 2ul | 1.5ul | 9ul | 1ul |
Template | 10x buffer | CES22k probes #4 (177nM) | H2O | ||
5 | Hues6 gDNA(113ng/ul) | 5ul | 1.5ul | 5ul | 2ul |
6 | Hues6 cDNA(155ng/ul) | 0.7ul | 1.5ul | 5ul | 6.3ul |
7 | H1 gDNA(222ng/ul) | 3.6ul | 1.5ul | 5ul | 3ul |
8 | H1 cDNA(48ng/ul) | 3ul | 1.5ul | 5ul | 5ul |
9 | H2 gDNA(200ng/ul) | 4ul | 1.5ul | 5ul | 2ul |
10 | H2 cDNA(35ng/ul) | 3ul | 1.5ul | 5ul | 5ul |
11 | BJ cDNA(18ng/ul) | 5ul | 1.5ul | 5ul | 2ul |
94C 5min->65C 40h-> add 1.5ul SLN mix (1U/ul stoffel, 1U/ul AmpLigase, 100uM dNTP) -> 65C 1h -> 40 cycles of (94C 2min -> 65C 15min) -> 37C 1min -> add 1.5ul of exo mix (Exo I, Exo III, T7 exo)->37c 1 hour -> 95C 5min -> 4C hold.
Note: I decided to terminate this experiment after the discussion with Billy regarding to some new modification of the protocol. I took the tubes out from the thermocycler prior to adding SLN mix, and stored them at -20C.
2007-12-11: I restarted the reactions with a modified protocol (use 60C instead of 65C).
94C 5min->60C 46h-> add 1.5ul SLN mix (2U/ul stoffel, 1U/ul AmpLigase, 200nM dNTP) -> 60C 1h -> 40 cycles of (94C 2min -> 650C 15min) -> 30C 1min -> add 1.5ul of exo mix (Exo I, Exo III, T7 exo)->37c 1 hour -> 95C 5min -> 4C hold.