Paris/October 4

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Revision as of 15:13, 4 October 2007 by Landrain (Talk | contribs)

Contents

Minipreps

  • from S49 = pKD46
  • pY-2P-IntC

Transformation of W121 and FR781 with pKD46

Houston we got a problem, the upstream construction for wanner lose the Chloramphenicol resistance

[Frt-CmR-Frt-pTet>>lox71-gfpTripart] (L58)

- First, we got an abnormally short length for the PCR with the wanner primers (about 1,7kb instead of 2,1kb; see October 2)
- Then i did a PCR on the same plasmid (L58) with the primers O18&O19. We got the same pattern of length (1,7kb and another one at about 3,5kb)
- To investigate the possibility to have lost 400kb in the CmR gene leading to the loss of resistence to Cm for the strain, i put some clones of L58 on plates with either Ampicilline or Chloramphenicol (10µg/mL).


> the clones grew on Amp but not on Cm...

it confirms the hypothesis of a loss inside the CmR gene.


> of course the results for the sequencing will give us the last word. They should come on Friday in late afternoon. We called the sequencing facility.

Digestions

Ligations

Culture of S28, strain having the [Frt-CmR-Frt] contruction