IGEM:Groningen/Notebook/iGEM 2011/2011/07/14: Difference between revisions
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== | ==14-07-11== | ||
<br> Today, Digestion, DNA clean up with High Pure PCR purification kit of Roche, ligation, transformation to create transformants <br> with: PBAD-cI-LVA-DT, PhybB-cI-LVA-DT and PBAD-RBS-GFP-DT | |||
<br> Also, colony 6 of LasR-LVA (yesterday) grew on LB with ampicillin. Plasmid prep, glycerol stock and sample for sequencing were made. | |||
<br> Digestion: | |||
<br> cI-LVA-DT colony 3 | |||
<br> 2μl vector | |||
<br> 1μl EcoR1 | |||
<br> 1μl XbaI | |||
<br> 3μl Fast digest buffer | |||
<br> 1μl Fast AP | |||
<br> 22μl MQ | |||
<br> | |||
<br> RBS-GFP-DT | |||
<br> 3μl vector | |||
<br> 1μl EcoR1 | |||
<br> 1μl XbaI | |||
<br> 3μl Fast digest buffer | |||
<br> 1μl Fast AP | |||
<br> 21μl MQ | |||
<br> | |||
<br> pBAD | |||
<br> 10μl pBAD | |||
<br> 1μl EcoR1 | |||
<br> 1μl SpeI | |||
<br> 2μl Fast digest buffer | |||
<br> 6μl MQ | |||
<br> | |||
<br> PhybB | |||
<br> 3μl PhybB | |||
<br> 1μl EcoR1 | |||
<br> 1μl SpeI | |||
<br> 2μl Fast digest buffer | |||
<br> 13μl MQ | |||
<br> Incubate the samples at 37°C for 30 min. | |||
<br> Do a DNA purification with the PCR purification kit from Roche | |||
<br> Ligation: | |||
<br> | |||
<br> PBAD-cI-LVA-DT colony 3 | |||
<br> 8.5μl vector | |||
<br> 6.1μl insert | |||
<br> 1μl T4 DNA ligase | |||
<br> 2μl T4 DNA ligase buffer | |||
<br> 2.4μl MQ | |||
<br> | |||
<br> PhybB-cI-LVA-DT colony 3 | |||
<br> 8.5μl vector | |||
<br> 4.1μl insert | |||
<br> 1μl T4 DNA ligase | |||
<br> 2μl T4 DNA ligase buffer | |||
<br> 4.4μl MQ | |||
<br> | |||
<br> PBAD-RBS-GFP-DT | |||
<br> 8.5μl vector | |||
<br> 6.3μl insert | |||
<br> 1μl T4 DNA ligase | |||
<br> 2μl T4 DNA ligase buffer | |||
<br> 2.2μl MQ | |||
<br> | |||
<br>Self ligation: | |||
<br> 8.5μl vector | |||
<br> 1μl T4 DNA ligase | |||
<br> 2μl T4 DNA ligase buffer | |||
<br> 8.5μl MQ | |||
<br> | |||
<br> Ligate at room temperature for 30 min. | |||
<br> | |||
<br>Transform as usual (see 12th of July) | |||
<br> | |||
<br> Plasmid prep of overnight culture construct: LasR-LVA-DT (plasmid backbone with ampicillin resistance) | |||
<br> Elute in 100μl MQ | |||
<br> Use 10μl for gel electrophoresis to check wether a plasmid is present in the sample. Plasmid was seen on a 1% agarose gel with <br>TBE (with the help of gelred) | |||
<br> Nanodrop result: 67.2 ng/μl. | |||
<br> Send for sequencing: 5μl Sample + 5μl primer of 5μM. | |||
<br> Glycerol stock was made: 250μl Glycerol 85% + 750μl bacterial culture (overnight culture) | |||
<br> | |||
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Revision as of 10:18, 14 July 2011
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14-07-11
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