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

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(New page: ==Exp.1 Received the Agilent library today, start to make probes== PCR (with NEB Taq master mix) x1 x32 E55k1(20nM) 0.1ul ...)
 
 
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==Exp.1 Received the Agilent library today, start to make probes==
==Exp.1 Start another round of E55kv1 probe production==
PCR (with NEB Taq master mix)
PCR (with NEB Taq master mix)
                               x1          x32      
                               x1          x96      
         E55k1(20nM)        0.1ul      3.2ul    
         E55k1(20nM)        0.1ul      9.6ul    
         2x Taq master mix    50ul      1600ul    
         2x Taq master mix    50ul      4800ul    
         100uM pAP1V6U      0.3ul       9.6ul    
         100uM pAP1V6U      0.3ul     28.8ul    
         100uM phosAP2V6    0.3ul       9.6ul    
         100uM phosAP2V6    0.3ul     28.8ul    
         H2O                49.3ul      1600ul    
         H2O                49.3ul      4800ul    


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.