IGEM:Paris Bettencourt 2012/Notebooks/Semantic group/day by day//2012/10/22: Difference between revisions
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==Cloning Kan** into pSB1C3== | ==Cloning Kan** into pSB1C3== | ||
Fail after gel purification. | |||
==Preparation of the Tecan experiment with Kan**== | |||
Since the sequence of QCM are good (we have a Kan gene with 2 amber mutation), we can prepare the Tecan experiment to quantify the leakiness of the system. | |||
===Double transformation=== | |||
We to double transform this new Kan** gene with either supD or RFP : | |||
I transformed in 20µL of MG1655 competent cells, 1.5µL of the miniprep of Kan** (From S1-1) and 1.5µL of pSC011 (supD) in triplicate. I did the same but with pSB1C3::RFP instead of supD. | |||
I also transformed this 3 plasmids alone, to test them. | |||
I have a negative control, which is only cell, without plasmid. | |||
*30' on ice | |||
*45" 42°C | |||
*2' on ice | |||
* add 200µL of hot LB | |||
*1h20' at 37°C | |||
* plate 200µL. | |||
* incubate 37°C O/N. | |||
All plates excepts single plasmid control are plated on Cm + Amp. Controls are plated on their right antibiotic (Cm or Amp). | |||
Revision as of 13:52, 22 October 2012
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Cloning Kan** into pSB1C3Fail after gel purification. Preparation of the Tecan experiment with Kan**Since the sequence of QCM are good (we have a Kan gene with 2 amber mutation), we can prepare the Tecan experiment to quantify the leakiness of the system. Double transformationWe to double transform this new Kan** gene with either supD or RFP : I transformed in 20µL of MG1655 competent cells, 1.5µL of the miniprep of Kan** (From S1-1) and 1.5µL of pSC011 (supD) in triplicate. I did the same but with pSB1C3::RFP instead of supD. I also transformed this 3 plasmids alone, to test them. I have a negative control, which is only cell, without plasmid.
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