Paris/October 3
From 2007.igem.org
Contents |
Sequencing reactions
In order to verify the quality of the ligation reaction products (of Sep. 18), the following sequencing reactions are being performed
TL_L44.1+O18 TL_L44.1+O19 TL_L44.2+O18 TL_L44.2+O19 TL_L45.1+O18 TL_L45.1+O19 TL_L45.2+O18 TL_L45.2+O19 TL_L46.1+O18 TL_L46.1+O19 TL_L47.1+O18 TL_L47.1+O19 TL_L53.7+O18 TL_L53.7+O19 TL_L53.7+O26 TL_L54.3+O18 TL_L54.3+O19 TL_L54.3+O26 TL_L54.4+O18 TL_L54.4+O19 TL_L54.4+O26 TL_L56.3+O18 TL_L56.3+O19 TL_L56.3+O25 TL_L57.3+O18 TL_L57.3+O19 TL_L57.3+O25 TL_L57.4+O18 TL_L57.4+O19 TL_L57.4+O25 TL_L58.5+O18 TL_L58.5+O19 TL_L58.7+O18 TL_L58.7+O19 TL_L59.6+O18 TL_L59.6+O19 TL_L59.6+O40 TL_L63.1+O18 TL_L63.1+O19 TL_L63.2+O18 TL_L63.2+O19 TL_L64.1+O18 TL_L64.1+O19 TL_L64.1+O25 TL_L64.2+O18 TL_L64.2+O19 TL_L64.2+O25
Transformation of W121 and FR781 with pKD46 didn't work
We got no clones. After reflexion, it appeared that the miniprep of pKD46 i did came from a 37°C culture, and pKD46 is thermosensible, the explanation is just that i transformed bacteria with no plasmid.
i launched a new culture of S49 at 30°C this time.
Culture for miniprep of S49 having the pKD46 plamid
with 5mL of Amp-LB at 30°C.
Culture for miniprep of a strain (S51) having the plasmid pY-2P-IntC
this plasmid will help us to integrate the [pTet>>lox71-GFPtripart-lox66-mRFP] construction into IntC site on the bacteria chromosome. This construction will permit to undergo some recombination rate experiments on the future strain.
The wanner PCR didn't give the right size, exploration for explanation
We got 1,7kb instead of 2,1kb.
- new PCR with VF/VR2 and homology site primers in order to compare size.
- 20µL mix 2X taq
- 16µL H2O
- 3µL plasmid (L58.5 and L58.7)
- 0,5µL*2 Oligo (O18&O19)
- We took the L58 clones from Amp Plate and tranfer them to Chloramphenicol Plate.
we have the hypothesis that a part of the chloramphenicol gene has been deleted.
