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===Exp.2 Probe prep. for the CES22k set on two new libraries=== PCR (with JumpStart Taq) x1 x16x2 template(100nM) 0.05ul 2x0.8ul 10x ABI buffer 10ul 320ul 25mM MgCl2 6ul 192ul 10mM dNTP 2ul 64ul 100uM pAP1V6U 0.3ul 9.6ul 100uM phosAP2V6 0.3ul 9.6ul JumpStart Taq 1ul 32ul H2O 80ul 2560ul 94C 3min -> 18 cycles of (94C 45sec -> 58C 2min -> 72C 1min) -> 72C 3min -> 15C hold Purify each amplicons with Bioneer 4 columns. Yield: *CES22k #4: 63ng/ul x 120ul *CES22k #5: 62ng/ul x 120ul Lambda exo digestion: to 120ul DNA, add 15ul 10X lambda exo buffer, 8ul lambda exo, 7ul H2O, 37C 4h, 75C 15min, add 10ul USER enzyme, 37C 2h. Purified 110ul of #4 and 40ul of #5 with Bioneer column, very little DNA was recovered. Ethanol precipitation the remaining DNA, resuspended in 40ul (#4) and 125ul(#5) ddH2O. Dpn II & USER digestion: Add 5ul/15 10X Dpn II buffer, 2ul/6ul RE-DpnIIn2s guide oligo, 95C 10min, terminate the PCR program and let the reactions cool down in 15min, add 4ul/10ul Dpn II, 2ul/8ul USER, 37C 4h, 75C 10min. Purified 55ul of #4 and 100ul of #5 with MinElute columns. Again I lost most DNA, only ~80ng was recovered for each sample. I proceed to PAGE purification on the unpurified #5.
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