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 used digestion by P and S (since LovTAP contains 2 P sites and Trp op contains 2 S sites). For reaction, incubated at 37°C (INCUB37) for about 1h30.

Gel

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

This time we are certain that the double-transformation didn't work in the Trp-mutated strains: from the digestion assay we could conclude that one of the strains contained only the LovTap plasmid, while the other one contained only the RO1. We will redo the transformations this weekend.

Results of the plate-reader experiment

Graphic :

People in the lab

Heidi, Gab, Tu