Kun:LabNotes/ASE/2007-11-20
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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
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.