IGEM:Groningen/Notebook/iGEM 2011/2011/07/11: Difference between revisions
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== | ==11-7-11== | ||
<br>PCR clean up of the pBAD araC PCR products. | |||
<br>Digestion of pBAD araC, cI-LVA, LasR-LVA and the vectors pSB1A3+DT and pSB1A3 + RBS-GFP | |||
<br>Digestion | |||
<br>pBAD araC: | |||
<br>pBADaraC: 10μl | |||
<br> EcoRI: 1μl | |||
<br> SpeI: 1μl | |||
<br> Fast digest buffer: 2μl | |||
<br> MilliQ water: 6μl | |||
<br> | |||
<br>cI-LVA: | |||
<br>cI-LVA: 2μl | |||
<br> EcoRI: 1μl | |||
<br> SpeI: 1μl | |||
<br> Fast digest buffer: 2μl | |||
<br> MilliQ water: 14μl | |||
<br> | |||
<br>LasR-LVA: | |||
<br>LasR-LVA: 3μl | |||
<br> EcoRI: 1μl | |||
<br> SpeI: 1μl | |||
<br> Fast digest buffer: 2μl | |||
<br> MilliQ water: 13μl | |||
<br> | |||
<br>pSB1A3+DT: | |||
<br>pSB1A3+DT: 3μl | |||
<br> EcoRI: 1μl | |||
<br> SpeI: 1μl | |||
<br> Fast digest buffer: 2μl | |||
<br> Fast alkaline phosphatase: 1μl | |||
<br> MilliQ water: 22μl | |||
<br> | |||
<br>pSB1A3+RBS-GFP: | |||
<br>pSB1A3+RBS-GFP: 3μl | |||
<br> EcoRI: 1μl | |||
<br> SpeI: 1μl | |||
<br> Fast digest buffer: 2μl | |||
<br> Fast alkaline phosphatase: 1μl | |||
<br> MilliQ water: 22μl | |||
<br> | |||
<br> Incubate at 37 °C for 30 minutes | |||
<br> Do after the digestion a DNA clean up with the High Pure PCR purification kit | |||
<br> Ligation | |||
<br> Calculate with the ligation calculator how much ng DNA is needed for your ligation and then calculate how many microliters this is. | |||
<br>cI-LVA in pSB1A3-DT | |||
<br> cI-LVA: 7.8 μl | |||
<br> pSB1A3-DT: 8.5μl | |||
<br> T4 DNA ligase buffer: 2μl | |||
<br> T4 DNA ligase: 1μl | |||
<br> MilliQ water: 0.7μl | |||
<br> | |||
<br>LasR-LVA in pSB1A3-DT | |||
<br> LasR-LVA: 6.1 μl | |||
<br> pSB1A3-DT: 8.5μl | |||
<br> T4 DNA ligase buffer: 2μl | |||
<br> T4 DNA ligase: 1μl | |||
<br> MilliQ water: 2.4μl | |||
<br> | |||
<br>pBAD araC in pSB1A3+RBS-GFP | |||
<br> pBAD araC: 7.8 μl | |||
<br> pSB1A3+ RBS-GFP: 8.5μl | |||
<br> T4 DNA ligase buffer: 2μl | |||
<br> T4 DNA ligase: 1μl | |||
<br> MilliQ water: 0.7μl | |||
<br> | |||
<br>pSB1A3 self ligation control | |||
<br> pSB1A3-DT: 8.5μl | |||
<br> T4 DNA ligase buffer: 2μl | |||
<br> T4 DNA ligase: 1μl | |||
<br> MilliQ water: 8.5μl | |||
<br> | |||
<br>pSB1A3+RBS-GFP | |||
<br> pSB1A3+RBS-GFP: 8.5μl | |||
<br> T4 DNA ligase buffer: 2μl | |||
<br> T4 DNA ligase: 1μl | |||
<br> MilliQ water: 8.5μl | |||
<br> | |||
<br> Incubate for 30 minutes at roomtemperature. | |||
<br> Transformation: | |||
<br> - Add to 40μl competent cells 10μl DNA ligation mixture | |||
<br> - Incubate 30 minutes on ice | |||
<br> - Do the heatshock: incubate 45 seconds at 42°C | |||
<br> - Place the tubes on ice for 2 minutes and then pipette 1 ml of LB medium + 25mM glucose | |||
<br> - Incubate the tubes for 1 hour (with maximum of 1.5h) at 37°C | |||
<br> - Pellet the cells and resuspend them in 100μl medium | |||
<br> - Plate 90μl and 10μl of the cells on LB+ampicillin plates | |||
<br> - Incubate overnight at 37°C | |||
<br> | |||
Revision as of 06:53, 11 July 2011
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11-7-11
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