EPF-Lausanne/31 August 2009

  

31 August 2009

Wet Lab
Digestion

The Klenow fragments that have been amplified by PCR (after the Klenow reaction) will be digested with SpeI. We have to check if it is indeed the TrpO (presence of the two S sites).

Results of the overweekend cultures

The idea was to find a new medium without any TRY on it, but on which the cells can still grow.

The minimal media + arginine didn't show any result (nothing grew).

The tubes induced by Try didn't change colour over the week-end. The two that were already red are still red and the others didn't change colour.

RO1 construction


Digestion of the concentrated Klenow PCR. After digestion, both gel extraction and purification kit will be tried.

We did a gel to check the digestion assay of TrpO and the samples from Saturday. TrpO, BB5, RO2#8 and the double transformation seems okay. The double constructs didn't work, for both clones (it seems not to have included the BB, all patterns are looking the same as the positive RO2 control).

Transformation of the following plasmids from kit plate n°1 have been transformed in DH5a : I13602 (on pSB1A2) and J04450 (on pSB1C3). The first plasmid is to check if DH5a has endogenous TetR gene (if it doesn't - as supposed - then the bacterium will express CFP, no matter if we had ATC or not. ATC needs to have the TetR protein to bind to and have an effect). The second plasmid is to have a test both on IPTG induction (on LacI as in our construction) and also to have a positive control for RFP.

The host vector (I13507) containing RFP has been digested with E and X. After incubation, purification using the purelink kit.

Gel extraction of the TrpO digestion products, using the Giagen gel extraction kit protocol.

Ligation of RO1 overnight.

People in the lab
Basile

 