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Kun:LabNotes/ExonomeSeq/2007-12-17
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==Exp.1 Start another round of E55kv1 probe production== PCR (with NEB Taq master mix) x1 x96 E55k1(20nM) 0.1ul 9.6ul 2x Taq master mix 50ul 4800ul 100uM pAP1V6U 0.3ul 28.8ul 100uM phosAP2V6 0.3ul 28.8ul H2O 49.3ul 4800ul 94C 3min -> 18 cycles of (94C 45sec -> 58C 2min -> 72C 1min) -> 72C 3min -> 15C hold Ethanol precipitation. <b>I put everything in a 50-ml tube for centrifugation. The tube broke during spinning. And I lost ~30% of the sample. The remaining sample contains some debris. After resuspending with 200ul ddH2O, I filtered the solution with NanoSep column. </b> The Nanodrop read is 4.3ug/ul, but it might not be accurate. I did lambda exo digestion on half of the DNA: 100ul DNA + 60ul H2O + 20ul 10X buffer + 20ul lambda exo -> 37C 8h -> 85C 20min -> 4C hold. Purified the digested DNA with two MinElute columns, eluted with 30ul EB buffer per column, added water to 100ul. Measured concentration with Nanodrop: 34ng/ul. Added 8ul USER enzyme, 37C 4h. Add 60ul H2O, 20ul 10X Dpn II buffer, 10ul 100um RE-DpnIIs buffer -> 95C 10min -> stop the program -> wait for 15min -> 37C 1min -> add 10ul Dpn II, 5ul USER -> 37C 6h -> 75C 10min -> 37C hold. [[Image:2008-01-04-E55kv1-probe-PAGE0-purification.png|600px]] <b> Seeing a band at ~200bp in the unpurified digestion is quite surprising. Perhaps it is the DNA:Enzyme complex? If that's the case, doing purification certainly help. </b>
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