IGEM:Groningen/Notebook/iGEM 2011/2011/07/22: Difference between revisions
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== | ==22-07-11== | ||
<br> | |||
<br> Plates of transformants with LasR-LVA DT vector look good, since the self ligation plates don't have much colonies! | |||
<br> 10μl plate is empty on self ligation plate control! 90μl plate has a few colonies (5) | |||
<br> Colony PCR as usual | |||
<br> | |||
<br> Digestion PBAD, PhybB and vectors: cI-LVA and RBS-GFP | |||
<br> | |||
<br>PBAD: | |||
<br> 15μl PBAD | |||
<br> 1μl SpeI | |||
<br> 2μl Tango buffer | |||
<br> 2μ MQ water | |||
<br> | |||
<br>PhybB | |||
<br> 3μl PhybB | |||
<br> 1μl SpeI | |||
<br> 2μl Tango buffer | |||
<br> 14μl MQ water | |||
<br> | |||
<br> After 1h and 50 minutes: put 2.5μl Tango buffer+ 1μl EcoRI in these samples and digest for another 1h and 50 minm. | |||
<br> DNA clean up with kit of Roche. | |||
<br> | |||
<br> Ligation: | |||
<br> PBAD-cI-LVA-DT | |||
<br> 6μl PBAD | |||
<br> 8.5μl cI-LVA-DT vector | |||
<br> 2μl T4 DNA ligase buffer | |||
<br> 1μl T4 DNA ligase | |||
<br> 2.5μl MQ water | |||
<br> | |||
<br> PhybB-cI-LVA-DT | |||
<br> 6μl PhybB | |||
<br> 8.5μl cI-LVA-DT vector | |||
<br> 2μl T4 DNA ligase buffer | |||
<br> 1μl T4 DNA ligase | |||
<br> 2.5μl MQ water | |||
<br> | |||
<br> PBAD-RBS-GFP-DT | |||
<br> 6.2μl PhybB | |||
<br> 8.5μl cI-LVA-DT vector | |||
<br> 2μl T4 DNA ligase buffer | |||
<br> 1μl T4 DNA ligase | |||
<br> 2.3μl MQ water | |||
<br> | |||
<br> Ligate for 30 to 45 minutes | |||
<br> | |||
<br> Transformation as usual | |||
<br> | |||
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Revision as of 02:38, 22 July 2011
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