IGEM:Groningen/2009/Notebook/iGEM 2011/2011/07/08: Difference between revisions
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==*8th of July== | ==*8th of July== | ||
Colony PCR of transformants to check if any transformant contains the plasmid with cI-LVA and LasR-LVA. | <br>Colony PCR of transformants to check if any transformant contains the plasmid with cI-LVA and LasR-LVA. | ||
20μl total volume was used and the PCR was done with taq polymerase. | <br>20μl total volume was used and the PCR was done with taq polymerase. | ||
PCR program: | <br>PCR program: | ||
Denaturation: 95 °C for ten minutes | <br>Denaturation: 95 °C for ten minutes | ||
Cycle (33×): | <br>Cycle (33×): | ||
<br> Denaturation: 95°C for 30 seconds | |||
<br> Annealing: 60°C for 30 seconds | |||
<br> Extension: 72°C for 2 minutes | |||
Final extension: 72°C for ten minutes | <br>Final extension: 72°C for ten minutes | ||
Store at 4 °C infinite | <br>Store at 4 °C infinite | ||
<br> | |||
PCR with the new pBAD primers (dissolved and dilution made by me) | <br>PCR with the new pBAD primers (dissolved and dilution made by me) | ||
PCR was done in total colume of 50μl and with tag and Pfu polymerase (ratio 6:1) so Pfu can correct the mistakes that taq made | <br>PCR was done in total colume of 50μl and with tag and Pfu polymerase (ratio 6:1) so Pfu can correct the mistakes that taq made | ||
Denaturation: 94°C for ten minutes | <br>Denaturation: 94°C for ten minutes | ||
Cycle (30×) | <br>Cycle (30×) | ||
<br> Denaturation: 94°C for 30 seconds | |||
<br> Annealing: 62°C and 65°C for 30 seconds | |||
<br> Extension: 72°C for 2 minutes | |||
Final extension: 72°C for ten minutes | <br>Final extension: 72°C for ten minutes | ||
Store at 4 °C infinite | <br>Store at 4 °C infinite | ||
<br> | |||
Check them on agarose gel 1% (TBE). | <br>Check them on agarose gel 1% (TBE). | ||
After that: Clean up the pBAD samples and nanodrop the samples | <br>After that: Clean up the pBAD samples and nanodrop the samples | ||
Nanodrop results: | <br>Nanodrop results: | ||
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Revision as of 03:17, 8 July 2011
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*8th of July
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