IGEM:Paris Bettencourt 2012/Notebooks/Semantic group/day by day//2012/10/22

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Current revision (16:52, 22 October 2012) (view source)
(Cloning Kan** into pSB1C3)
 
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==Cloning Kan** into pSB1C3==
==Cloning Kan** into pSB1C3==
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Fail after gel purification.
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==Preparation of the Tecan experiment with Kan**==
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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.
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===Double transformation===
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We to double transform this new Kan** gene with either supD or RFP :
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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.
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I also transformed this 3 plasmids alone, to test them.
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I have a negative control, which is only cell, without plasmid.
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*30' on ice
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*45" 42°C
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*2' on ice
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* add 200µL of hot LB
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*1h20' at 37°C
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* plate 200µL.
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* incubate 37°C O/N.
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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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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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