IGEM:Groningen/Notebook/iGEM 2011/2011/08/09: Difference between revisions
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== | ==9-8-11== | ||
<br> | |||
<br> Colony PCR of yesterday's transformation. Analyse it on a 1% agarosegel | |||
<br> Overnightculture of RBS-GFP-DT grew. Plasmid prep of RBS-GFP-DT overnight culture | |||
<br> Measuring DNA concentration with the ND1000 nanodrop: 32.6ng/μl | |||
<br> | |||
<br> Digestion of pSB1C3+ PBAD/araC, RBS-GFP-DT vector and PBAD/araC | |||
<br> RBS-GFP-DT vector: | |||
<br> 5μl vector | |||
<br> 1μl EcoRI | |||
<br> 1μl XbaI | |||
<br> 1μl FastAP | |||
<br> 3μl FD buffer | |||
<br> 19μl MQ water | |||
<br> | |||
<br> PBAD/araC: | |||
<br> 5μlPBAD/araC | |||
<br> 1μl EcoRI | |||
<br> 1μl SpeI | |||
<br> 2μl FD buffer | |||
<br> 11μl MQ water | |||
<br> | |||
<br> pSB1C3+PBAD/araC | |||
<br> 2μl vector | |||
<br> 2μl PBAD/araC | |||
<br> 1μl EcoRI | |||
<br> 1μl PstI | |||
<br> 2μl FD buffer | |||
<br> 12μl MQ water | |||
<br> Digest for 1h at 37 degrees | |||
<br> DNA clean up with PCR clean up kit | |||
<br> Ligation: | |||
<br> PBAD/araC-RBS-GFP-DT | |||
<br> 8.5μl vector | |||
<br> 5.6μl PBAD/araC | |||
<br> 1μl T4 DNA ligase | |||
<br> 2μl T4 DNA ligase buffer | |||
<br> 2.9μl MQ water | |||
<br> | |||
<br> Self ligation RBS-GFP-DT | |||
<br> 8.5μl vector | |||
<br> 1μl T4 DNA ligase | |||
<br> 2μl T4 DNA ligase buffer | |||
<br> 8.5μl MQ water | |||
<br> | |||
<br> pSB1C3+PBAD/araC | |||
<br> In the DNA clean up step, the DNA was eluted in 17μl for direct use in ligation | |||
<br> so: | |||
<br> 17μl pSB1C3+PBAD/araC * EcoRI*PstI purified | |||
<br> 1μl T4 DNA ligase | |||
<br> 2μl T4 DNA ligase buffer | |||
<br> | |||
<br> Transformation: | |||
<br> Add to 40μl competent cells 10μl ligation mixture | |||
<br> Incubate for 30 min on ice | |||
<br> Heat shock: 45s at 42 degrees | |||
<br> Incubate cells on ice for 2 min | |||
<br> Add 1ml LB medium+ 25mM glucose | |||
<br> Incubate cells for 1h/1.5h at 37 degrees | |||
<br> Spin the cells down, resuspend them in 100μl LB medium and plate 90μl and 10μl out on the plates | |||
<br> Put the plates in the stove at 37 degrees overnight | |||
<br> | |||
Revision as of 05:22, 9 August 2011
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