IGEM:Groningen/Notebook/iGEM 2011/2011/08/29: Difference between revisions
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== | ==29-8-11== | ||
<br> Cloning PBAD-RBS-GFP (in 2 different ways), PBAD-cI-LVA and PBAD-LasR-LVA | |||
<br> Calculations were made with the Ligation calculator based on DNA concentrations according to the nanodrop | |||
<br> Digestion: | |||
<br> PBADaraC | |||
<br> 4μl insert (PBADaraC) | |||
<br> 1μl EcoRI | |||
<br> 1μl SpeI | |||
<br> 2μl FD buffer | |||
<br> 12μl MQ water | |||
<br> | |||
<br> RBS-GFP-DT vector: | |||
<br> 3μl vector | |||
<br> 1μl EcoRI | |||
<br> 1μl XbaI | |||
<br> 3μl FD buffer | |||
<br> 1μl FastAP | |||
<br> 21μl MQ | |||
<br> | |||
<br> RBS-GFP-DT | |||
<br> 14μl ´insert´ | |||
<br> 1μl XbaI | |||
<br> 1μl PstI | |||
<br> 2μl FD buffer | |||
<br> 2μl MQ water | |||
<br> | |||
<br> PBADaraC vector | |||
<br> 4μl vector | |||
<br> 1μl SpeI | |||
<br> 1μl PstI | |||
<br> 3μl FD buffer | |||
<br> 1μl FastAP | |||
<br> 20μl MQ water | |||
<br> | |||
<br> cI-LVA | |||
<br> 7μl insert | |||
<br> 1μl XbaI | |||
<br> 1μl PstI | |||
<br> 2μl FD buffer | |||
<br> 9μl MQ water | |||
<br> | |||
<br> LasR-LVA | |||
<br> 12μl insert | |||
<br> 1μl XbaI | |||
<br> 1μl PstI | |||
<br> 2μl FD buffer | |||
<br> 4μl MQ water | |||
<br> | |||
<br> Incubate the samples for 1h at 37 °C. | |||
<br> | |||
<br> DNA clean up with the High Pure PCR Purification Kit of Roche | |||
<br> | |||
<br> Ligation: | |||
<br> PBADaraC-RBS-GFP-DT | |||
<br> 6μl PBADaraC | |||
<br> 8.5μl vector RBS-GFP-DT | |||
<br> 2μl T4 DNA ligase buffer | |||
<br> 1μl T4 DNA ligase | |||
<br> 2.5μl MQ water | |||
<br> | |||
<br> PBAD-RBS-GFP-DT | |||
<br> 8.1μl RBS GFP ('insert') | |||
<br> 8.5μl PBADaraCvector | |||
<br> 2μl T4 DNA ligase buffer | |||
<br> 1μl T4 DNA ligase | |||
<br> 0.4μl MQ water | |||
<br> | |||
<br> PBADaraC-cI-LVA | |||
<br> 8.2μl cI-LVA | |||
<br> 8.5μl PBADaraCvector | |||
<br> 2μl T4 DNA ligase buffer | |||
<br> 1μl T4 DNA ligase | |||
<br> 0.3μl MQ water | |||
<br> | |||
<br> PBADaraC-LasR-LVA | |||
<br> 8.1μl LasR-LVA | |||
<br> 8.5μl PBADaraCvector | |||
<br> 2μl T4 DNA ligase buffer | |||
<br> 1μl T4 DNA ligase | |||
<br> 0.4μl MQ water | |||
<br> | |||
<br> Incubate the samples for 30 to 40 minutes at roomtemperature | |||
<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 03:37, 29 August 2011
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