Kun:LabNotes/ASE/2007-12-9

From ZhangLabWiki
Jump to navigation Jump to search

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.