Kun:LabNotes/ExonomeSeq/2007-12-12
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Exp.1 Received the Agilent library today, start to make probes[edit]
PCR (with iTaq)
x1 x32 x1 x32 E55k1(20nM) 0.1ul 3.2ul CES22k #4/#5 0.05ul 1.6ul 10x Sigma buffer 10ul 32ul 10ul 32ul 10mM dNTP 2ul 64ul 2ul 64ul 100uM pAP1V6U 0.3ul 9.6ul 0.3ul 9.6ul 100uM phosAP2V6 0.3ul 9.6ul 0.3ul 9.6ul 50X SYBG I 0.8ul 25.6ul 0.8ul 25.6ul iTaq 1ul 32ul 1ul 32ul H2O 85.5ul 2736ul 85.5ul 2736ul
94C 3min -> 20 cycles of (94C 45sec -> 58C 2min -> 72C 1min) -> 72C 3min -> 15C hold
Ethanol precipitation, resuspend in 190ul dH2O.
File:2007-12-13-E55kv1-lib-PCR.png Gel quantification: E55kv1: 17ng/ul x 190ul = 3.23ug CES22k#4: 26ng/ul x 190ul = 4.94ug CES22k#5: 25ng/ul x 190ul = 4.75ug
Lambda exo digestion: add 20ul 10X lambda exo buffer to 170ul amplicons, 10ul lambda exo, 37C 4h, 75C 15min, ethanol precipitation, resuspend in 160ul dH2O.
Dpn II & USER digestion: Add 20ul 10X Dpn II buffer, 10ul RE-DpnIIn2s guide oligo, 95C 10min, terminate the PCR program and let the reactions cool down in 15min, add 10ul Dpn II, 5ul USER, 37C 16h, 75C 10min.