IGEM:Groningen/Notebook/iGEM 2011/2011/07/11

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11-7-11
PCR clean up of the pBAD araC PCR products. Digestion of pBAD araC, cI-LVA, LasR-LVA and the vectors pSB1A3+DT and pSB1A3 + RBS-GFP Digestion pBAD araC: pBADaraC: 10μl EcoRI: 1μl SpeI: 1μl Fast digest buffer: 2μl MilliQ water: 6μl cI-LVA: cI-LVA: 2μl EcoRI: 1μl SpeI: 1μl Fast digest buffer: 2μl MilliQ water: 14μl LasR-LVA: LasR-LVA: 3μl EcoRI: 1μl SpeI: 1μl Fast digest buffer: 2μl MilliQ water: 13μl pSB1A3+DT: pSB1A3+DT: 3μl EcoRI: 1μl SpeI: 1μl Fast digest buffer: 2μl Fast alkaline phosphatase: 1μl MilliQ water: 22μl pSB1A3+RBS-GFP: pSB1A3+RBS-GFP: 3μl EcoRI: 1μl SpeI: 1μl Fast digest buffer: 2μl Fast alkaline phosphatase: 1μl MilliQ water: 22μl Incubate at 37 °C for 30 minutes Do after the digestion a DNA clean up with the High Pure PCR purification kit Ligation Calculate with the ligation calculator how much ng DNA is needed for your ligation and then calculate how many microliters this is. cI-LVA in pSB1A3-DT cI-LVA: 7.8 μl pSB1A3-DT: 8.5μl T4 DNA ligase buffer: 2μl T4 DNA ligase: 1μl MilliQ water: 0.7μl LasR-LVA in pSB1A3-DT LasR-LVA: 6.1 μl pSB1A3-DT: 8.5μl T4 DNA ligase buffer: 2μl T4 DNA ligase: 1μl MilliQ water: 2.4μl pBAD araC in pSB1A3+RBS-GFP pBAD araC: 7.8 μl pSB1A3+ RBS-GFP: 8.5μl T4 DNA ligase buffer: 2μl T4 DNA ligase: 1μl MilliQ water: 0.7μl pSB1A3 self ligation control pSB1A3-DT: 8.5μl T4 DNA ligase buffer: 2μl T4 DNA ligase: 1μl MilliQ water: 8.5μl pSB1A3+RBS-GFP pSB1A3+RBS-GFP: 8.5μl T4 DNA ligase buffer: 2μl T4 DNA ligase: 1μl MilliQ water: 8.5μl Incubate for 30 minutes at roomtemperature. Transformation: - Add to 40μl competent cells 10μl DNA ligation mixture - Incubate 30 minutes on ice - Do the heatshock: incubate 45 seconds at 42°C - Place the tubes on ice for 2 minutes and then pipette 1 ml of LB medium + 25mM glucose - Incubate the tubes for 1 hour (with maximum of 1.5h) at 37°C - Pellet the cells and resuspend them in 100μl medium - Plate 90μl and 10μl of the cells on LB+ampicillin plates - Incubate overnight at 37°C


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