Kun:LabNotes/ExonomeSeq/2007-12-17: Difference between revisions

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94C 3min -> 18 cycles of (94C 45sec -> 58C 2min -> 72C 1min) -> 72C 3min -> 15C hold
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>

Latest revision as of 01:52, 5 January 2008

Exp.1 Start another round of E55kv1 probe production[edit]

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

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.