EPF-Lausanne/8 October 2009

Wet Lab

Colony PCR

On the same double transformants -> 2 min extension.

Gel

Here the 2nd strains seems to have a double transformant. But we let the gel overnight so there is no BrEth any more, we had to take a time of exposure of 38.2 s to see something. We will do a nicer gel this afternoon.

Preparation of the time-course experiment

The idea is to look at the evolution of fluorescence over the time, for different time of exposition on the light.

7 ml of LB + 1 ml of DH5a RO2.4 + BB1 #3 overnight culture.

5 conditions :

- + light + IPTG - Trp

- + light - IPTG - Trp

- - light + IPTG - Trp

- - light - IPTG + Trp

- - light - IPTG + Trp

- - light - IPTG - Trp

IPTG at 1 mM (100 mM sol. diluted 1/100). Trp diluted 1/25. Incubated in conditions at 37°C. Measures will be taken with plate reader every 30 minutes starting at 1h.

Miniprep

We did a miniprep of new strain double transformants to confirm gel results by digestion assay :

- RO1.1 + BB1 JRG 1046 (n°1,3,7)

- RO2.4 + BB1 JRG 1046 (n°1,3,6)

+ miniprep of DH5 a RO2.4 + BB1 #3 (strain that works) to have the 2 plasmids for subsequent transformations.

Digestion assay

For each clones, we use digestion by P and S (since LovTAP contains 2 P sites and Trp op contains 2 S sites). For reaction, incubate at 37°C (INCUB37). Digestion of about 1h30.

Gel

Once again to see the double transformants + of digestion assay.

Result ?? TO BE COMPLETED

People in the lab

Heidi, Gab, Tu