Kun:LabNotes/ExonomeSeq/2007-12-17: Difference between revisions
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==Exp.1 Start another round of E55kv1 probe production== | ==Exp.1 Start another round of E55kv1 probe production== | ||
PCR (with NEB Taq master mix) | PCR (with NEB Taq master mix) | ||
x1 | x1 x96 | ||
E55k1(20nM) 0.1ul | E55k1(20nM) 0.1ul 9.6ul | ||
2x Taq master mix 50ul | 2x Taq master mix 50ul 4800ul | ||
100uM pAP1V6U 0.3ul | 100uM pAP1V6U 0.3ul 28.8ul | ||
100uM phosAP2V6 0.3ul | 100uM phosAP2V6 0.3ul 28.8ul | ||
H2O 49.3ul | 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.