IGEM:Groningen/Notebook/iGEM 2011/2011/06/27: Difference between revisions
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== | ==27-6-11== | ||
<br> | |||
<br> Restriction analysis: | |||
<br> 2μl FD buffer | |||
<br> 0.5μl EcoRI | |||
<br> 0.5μl PstI | |||
<br> 7μl MQ water | |||
<br> 10μl mix+ 10μl sample | |||
<br> | |||
<br> Mix 2: | |||
<br> 8μl FD buffer | |||
<br> 2μl EcoRI | |||
<br> 2μl PstI | |||
<br> 28μl MQ water | |||
<br> 10μl mix+ 10μl sample | |||
<br> | |||
<br> Digestion vector: | |||
<br> 4μl FD buffer | |||
<br> 1μl EcoRI | |||
<br> 1μl PstI | |||
<br> 14μl MQ water | |||
<br> 10μl mix+ 10μl sample | |||
<br> Digestion insert, PBAD, PcI, PlasB: | |||
<br> 4μl FD buffer | |||
<br> 1μl EcoRI | |||
<br> 1μl PstI | |||
<br> 14μl MQ water | |||
<br> 10μl mix+ 10μl sample | |||
<br> Digest for 1h at 37 degrees | |||
<br> | |||
<br> Analyse PhybB-RBS-GFP-DT on 1% TBE agarose gel | |||
<br> | |||
<br> Ligate other digested DNA fragments with other promotors in vector with RBS-GFP-DT | |||
<br> 2μl T4 DNA ligase buffer | |||
<br> 1μl T4 DNA ligase | |||
<br> 5μl vector | |||
<br> 6μl insert | |||
<br> 6μl MQ water | |||
<br> | |||
<br> Ligate for 2hours at room temperature | |||
<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 | |||
Revision as of 03:47, 21 September 2011
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27-6-11
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