Paris/October 4
From 2007.igem.org
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.
Digestion reactions
| Digestion Products | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Number | Product Name | Matrix Name | Enzyme 1 | Enzyme 2 | Size | Description | |||
| D79 | L44.1 | Xba1 | Pst1 | ||||||
| D80 | L44.2 | Xba1 | Pst1 | ||||||
| D81 | L45.1 | Xba1 | Pst1 | ||||||
| D82 | L45.2 | Xba1 | Pst1 | ||||||
| D83 | L46.1 | Xba1 | Pst1 | ||||||
| D84 | L47.1 | Xba1 | Pst1 | ||||||
| D85 | L53.7 | EcoRI | SpeI | ||||||
| D86 | L54.3 | EcoRI | SpeI | ||||||
| D87 | L54.4 | EcoRI | SpeI | ||||||
| D88 | L56.3 | EcoRI | SpeI | ||||||
| D89 | L57.1 | EcoRI | SpeI | ||||||
| D90 | L57.3 | EcoRI | SpeI | ||||||
| D91 | MP14.1 | EcoRI | XbaI | ||||||
| D92 | MP14.2 | EcoRI | XbaI | ||||||
| D93 | MP14.1 | Spe1 | Pst1 | ||||||
| D94 | MP14.2 | Spe1 | Pst1 | ||||||
Ligations
| Ligations | ||||||
|---|---|---|---|---|---|---|
| Number | Insert | Insert Volume (µL) | Vector | Vector Volume (µL) | Comments | Number of colonies |
| C19 | D72 | 2 | ||||
| C20 | D73 | 2 | ||||
| C21 | D64 | 2 | ||||
| L65 | D56 | 10 | D72 | 2 | ||
| L66 | D75 | 10 | D73 | 2 | ||
| L67 | D59 | 10 | D64 | 2 | ||
Culture of S28, strain having the [Frt-CmR-Frt] contruction
In order to test the Chloramphenicol resistance of this strain.
Transformation of MP14.1 and MP14.2 in DH5α
In order to test the Chloramphenicol resistance given by MP14.
