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 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).



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