IGEM:Groningen/Notebook/iGEM 2011/2011/08/09

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