IGEM:Groningen/Notebook/iGEM 2011/2011/07/11: Difference between revisions
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<br>pSB1A3+DT: 3μl | <br>pSB1A3+DT: 3μl | ||
<br> EcoRI: 1μl | <br> EcoRI: 1μl | ||
<br> SpeI: 1μl | <br> SpeI: 1μl --> XbaI? | ||
<br> Fast digest buffer: 2μl | <br> Fast digest buffer: 2μl | ||
<br> Fast alkaline phosphatase: 1μl | <br> Fast alkaline phosphatase: 1μl | ||
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<br>pSB1A3+RBS-GFP: 3μl | <br>pSB1A3+RBS-GFP: 3μl | ||
<br> EcoRI: 1μl | <br> EcoRI: 1μl | ||
<br> SpeI: 1μl | <br> SpeI: 1μl --> XbaI? | ||
<br> Fast digest buffer: 2μl | <br> Fast digest buffer: 2μl | ||
<br> Fast alkaline phosphatase: 1μl | <br> Fast alkaline phosphatase: 1μl | ||
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<br> | <br> | ||
<br> Incubate at 37 °C for 30 minutes | <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> Do after the digestion a DNA clean up with the High Pure PCR purification kit --> how much MQ did you use for DNA elution? | ||
<br> Ligation | <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> Calculate with the ligation calculator how much ng DNA is needed for your ligation and then calculate how many microliters this is. | ||
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<br> - Add to 40μl competent cells 10μl DNA ligation mixture | <br> - Add to 40μl competent cells 10μl DNA ligation mixture | ||
<br> - Incubate 30 minutes on ice | <br> - Incubate 30 minutes on ice | ||
<br> - Do the heatshock: incubate | <br> - Do the heatshock: incubate 1 min at 42°C | ||
<br> - Place the tubes on ice for 2 minutes and then pipette 1 ml of LB medium + 25mM glucose | <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> - Incubate the tubes for 1 hour (with maximum of 1.5h) at 37°C |
Revision as of 02:45, 20 September 2011
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11-7-11
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