Paris/October 4

From 2007.igem.org

(Difference between revisions)
(Minipreps)
(Houston we got a problem, the upstream construction for wanner lose the Chloramphenicol resistance)
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== Houston we got a problem, the upstream construction for wanner lose the Chloramphenicol resistance ==
== Houston we got a problem, the upstream construction for wanner lose the Chloramphenicol resistance ==
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 +
[Frt-CmR-Frt-pTet>>lox66-gfpTripart] (L58)<br>
 +
<br>
 +
- First, we got an abnormally short length for the PCR with the wanner primers (about 1,7kb instead of 2,1kb; see October 2)<br>
 +
- 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)<br>
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- 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).<br>
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----> the clones grew on Amp but not on Cm... <br>
 +
it confirms the hypothesis of a loss inside the CmR gene.

Revision as of 12:30, 4 October 2007

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>>lox66-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.