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. 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.

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.

 File:2008-01-04-E55kv1-probe-PAGE0-purification.png