IGEM:Groningen/Notebook/iGEM 2011/2011/06/29: Difference between revisions
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== | ==29-6-11== | ||
<br> New lab plan: | |||
<br> Clone promotors upstream RBS-GFP-DT with a different approach | |||
<br> Clone cI-LVA, PhybB and LasR-LVA in pSB1C3 and whenever that is finished, clone upstream cI-LVA and LasR-LVA the PhybB <br> promotor | |||
<br> | |||
<br> Calculate with the ligation calculator how much of the insert is needed for a vector: insert ratio of 1:6 | |||
<br> | |||
<br> Digestion: | |||
<br> 3μl RBS-GFP-DT | |||
<br> 1μl EcoRI | |||
<br> 1μl XbaI | |||
<br> 2μl FD buffer | |||
<br> 13μl MQ water | |||
<br> insert: | |||
<br> 4μl insert | |||
<br> 1μl EcoRI | |||
<br> 1μl SpeI | |||
<br> 2μl FD buffer | |||
<br> 12μl MQ water | |||
<br> | |||
<br> Digestion one reaction: | |||
<br> 1μl pSB1C3 (50 ng) | |||
<br> 0.5μl insert | |||
<br> 0.5μl EcoRI | |||
<br> 0.5μl PstI | |||
<br> 2μl FD buffer | |||
<br> 15.5μl MQ water | |||
<br> | |||
<br> Digest for 1h at 37 degrees | |||
<br> | |||
<br> DNA clean up with High Pure PCR Purification Kit | |||
<br> | |||
<br> Ligation: | |||
<br> For the 1 digestion reaction: elute in the purification step in 17μl | |||
<br> Add: 2μl T4 DNA ligase buffer and 1μl T4 DNA ligase | |||
<br> | |||
<br> Ligation promotors upstream RBS-GFP-DT | |||
<br> 2μl T4 DNA ligase buffer | |||
<br> 1μl T4 DNA ligase | |||
<br> 8.5μl vector | |||
<br> 6μl insert | |||
<br> 2.5μl MQ water | |||
<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 | |||
<br> | |||
Revision as of 03:58, 21 September 2011
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