EPF-Lausanne/7 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

Prepared cells for time-course experiment:

- 7mL of fresh LB + 1mL of overnight cell culture (DH5 alpha RO2.4 + BB1 clone n.3)

- 5 different conditions:

         - +light +IPTG -Trp

         - +light -IPTG -Trp

         - -light +IPTG -Trp

         -  -light -IPTG +Trp

         -  -light -IPTG -Trp

The cells were put in the incubator at 37°C in their respective experimental conditions. Measurements of OD and fluorescence will be taken every 30 min starting at 1h of incubation, so that we have a time-course measurement. For the last sample, we will do a kinetic measurement overnight to see the decay of the RFP fluroescence.

Miniprep

We did a miniprep of the Trp-mutated strains that should be double transformants to extract the DNA and make a digestion assay in order to confirm the gel's results :

- RO1.1 + BB1 JRG 1046 (n°1,3,7)

- RO2.4 + BB1 JRG 1046 (n°1,3,6)

Also miniprepped DH5 alpha RO2.4+BB1 clone n.3 to extract the 2 working plasmids (LovTap and read-out 2), so that we can use this DNA directly for future 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

Results of the plate-reader experiment

Graphic :

People in the lab

Heidi, Gab, Tu