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==Exp.2 Probe prep for Cosmic3760, CpG28k and CES27k== PCR (with NEB Taq master mix) Cosmic3760 CpG28k CES27k x1 x25 Cosmic3760(20nM) 0.1ul 2.5ul CpgCES (20nM) 2.5ul 2.5ul 2x Taq master mix 50ul 1250ul 1250ul 1250ul 100uM AP1V41U 0.4ul 10ul 10ul pAP1V6U 10ul 100uM pAP2V4 0.4ul 10ul 10ul phosAP2V6 10ul H2O 49.3ul 1230ul 1230ul 1230ul 94C 3min -> 20 cycles of (94C 45sec -> 58C 2min -> 72C 1min) -> 72C 3min -> 15C hold Ethanol precipitation. Resuspended in 170ul dH2O, add 20ul 10X Lambda exo buffer, 10ul 5U/ul lambda exo, 37C 3h -> 90C 10min. Purify with Qiaquick columns. Yield: Cosmic3760=53ng/ul x 80ul; Cpg28k=33ng/ulx80ul; CES27k=33ng/ul x 80ul. Added 4ul USER enzyme, 37C 2h. Add 90ul H2O, 20ul 10X DpnII buffer, 10ul DpnIIV4/5ul DpnIIV4/5ul RE-DpnIIs, 94C 5min -> slowly cool down to 37C -> add 10ul Dpn II -> 37C 2h -> add 5ul USER -> 37C 2h -> 75C 10min -> 4C hold. I took 100ul Cosmic3760 and 100ul CpG28k out after 2-hour digestion. Did phenol/chloroform extraction, then PAGE purification. The palettes were resuspended in 10ul phosphorylation mix (1X T4PNK buffer, 1mM ATP, 1U/ul T4 PNK), 37C 10min. Then diluted to 20ul with ddH2O. Yield: *Cosmic3760: 6.2ng/ul => 194nM x 20ul *CpG: 3.1nM => 97nM x 20ul The remaining digestions were left until the reactions were completed. I heated the reactions at 95C for ~10min. Then did another round of PAGE on 100ul reactions on 01/26/08. Yield: *CES27k: 6.8ng/ul => 209nM x 20ul *Cosmic3760: 7.1ng/ul => 218nM x 20ul *Cpg28k: 3.4ng/ul => 104nM x 20ul
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