Kun:LabNotes/ASE/2007-11-20: Difference between revisions

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         RepliPhi phi-29      5ul
         RepliPhi phi-29      5ul
         30C 3h -> 85C 10min
         30C 3h -> 85C 10min
         Purify with Microcon YM-100
         Purify with Microcon YM-100 => 576ng/ul


==Exp. 2 Perform CES22k eSNP capture on PGP and hybrid samples==
==Exp. 2 Perform CES22k eSNP capture on PGP and hybrid samples==

Revision as of 06:50, 21 November 2007

Exp. 1 Perform MDA on the gDNA from BJ Fibroblast

       Template DNA(6ng/ul)  20ul
       10X RepliPhi buffer  2.5ul
       1mM N6               2.5ul
       95C 3min -> chill on ice -> add the reaction mix:    
       10X RepliPhi buffer 8.5ul
       25mM dNTP             4ul
       1mM N6              2.5ul
       H2O                  49ul
       100mM DTT             5ul
       RepliPhi phi-29       5ul
       30C 3h -> 85C 10min
       Purify with Microcon YM-100 => 576ng/ul

Exp. 2 Perform CES22k eSNP capture on PGP and hybrid samples

Template 10x buffer CES22k probes (277nM) H2O
GM20431 gDNA(347ng/ul) 2.3ul 1ul 5.4ul 0.3ul
GM20431 cDNA (121ng/ul) 0.4ul 1ul 5.4ul 2.2ul
GC1F cDNA(48ng/ul) 1ul 1ul 5.4ul 1.6ul
GC1EP cDNA(52ng/ul) 1ul 1ul 5.4ul 1.6ul
Hues6 gDNA(113ng/ul) 2.6ul 1ul 5.4ul 0ul
Hues6 cDNA(155ng/ul) 0.3ul 1ul 5.4ul 2.3ul
H1 gDNA(222ng/ul) 2.6ul 1ul 5.4ul 0ul
H1 cDNA(35ng/ul) 1.4ul 1ul 5.4ul 1.2ul
H2 gDNA(200ng/ul) 2.6ul 1ul 5.4ul 0ul
H2 cDNA(48ng/ul) 1ul 1ul 5.4ul 1.6ul
BJ MDA DNA(576ng/ul) 2.6ul 1ul 5.4ul 0ul
BJ cDNA(18ng/ul) 2.6ul 1ul 5.4ul 0ul

94C 5min->60C 18h-> add 0.5ul SLN mix (1U/ul stoffel, 0.5U/ul AmpLigase, 100nM dNTP) -> 60C 4h -> 15 cycles of (94C 1min -> 60C 1h) -> 60C 4h -> 37C 1min -> add 3ul of exo mix (Exo I, Exo III, T7 exo)->37c 1 hour -> 94C 5min -> 4C hold.