IGEM:Groningen/Notebook/iGEM 2011/2011/08/31: Difference between revisions
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== | ==31-8-11== | ||
<br> | |||
<br> Making schemes for this week: Cloning strategy | |||
<br> Today clone cI-LVA and LasR-LVA in pSB1C3-DT, but digest with X and P because the PCR products only contain the XbaI site. | |||
<br> This means that the DT will be excluded. This also means that an extra cloning step will be included to clone the double | |||
<br> terminator in the vector. | |||
<br> Tomorrow: Colony PCR of transformants, also clone PBAD vector in pSB1A3DT. | |||
<br> Friday: Colony PCR of PBAD in pSB1A3DT and if there were right transformants yesterday: plasmid prep, digestion cI-LVA and <br> LasR-LVA in vector and digest the double terminator PCR product, clean up, ligation, transformation. | |||
<br> | |||
<br> So today: | |||
<br> Digestion | |||
<br> pSB1C3DT | |||
<br> 3μl vector | |||
<br> 1μl XbaI | |||
<br> 1μl PstI | |||
<br> 3μl FD buffer | |||
<br> 1μl FastAP | |||
<br> 21μl MQ water | |||
<br> | |||
<br> cI-LVA | |||
<br> 10μl insert | |||
<br> 1μl XbaI | |||
<br> 1μl PstI | |||
<br> 2μl FD buffer | |||
<br> 6μl MQ water | |||
<br> | |||
<br> LasR-LVA | |||
<br> 11μl | |||
<br> 1μl XbaI | |||
<br> 1μl PstI | |||
<br> 2μl FD buffer | |||
<br> 5μl MQ water | |||
<br> | |||
<br> Incubate the samples at 37°C for 1h | |||
<br> | |||
<br> Clean up the DNA with the High Pure PCR purification kit | |||
<br> | |||
<br> Ligation: | |||
<br> | |||
<br> cI-LVA-pSB1C | |||
<br> 8.5μl vector pSB1C3-DT | |||
<br> 8.5μl insert | |||
<br> 2μl T4DNA ligase buffer | |||
<br> 1μl T4 DNA ligase | |||
<br> | |||
<br> LasR-LVA-pSB1C | |||
<br> 8.5μl vector pSB1C3-DT | |||
<br> 8.5μl insert | |||
<br> 2μl T4DNA ligase buffer | |||
<br> 1μl T4 DNA ligase | |||
<br> | |||
<br> Self ligation pSB1C3-DT | |||
<br> 8.5μl vector pSB1C3-DT | |||
<br> 8.5μl MQ water | |||
<br> 2μl T4DNA ligase buffer | |||
<br> 1μl T4 DNA ligase | |||
<br> | |||
<br> Incubate samples for 35 min. at room temperature | |||
<br> | |||
Revision as of 02:43, 31 August 2011
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