EPF-Lausanne/10 September 2009

Wet Lab

Colony PCR

We did a colony PCR of the 3 BB / Chl (A, B, C) that have grown (after an overnight + 1 day culture on a plate). It is strange they took so long to grow, so we hope it is not a contamination.

We took a couple of clones on each plate and used the Taq platinium protocol + colony file. We will run a gel to check if the clones are what we wanted.

Results of the culture

All the tubes grew (RO2 / RO1 / double transf.). The medium was only M9 (M9 + AA + thiamine) and not LB this time.

- RO1 #1 (red), #2 (red) which is very nice as it is in M9 (-TRP).

- RO2 #4 (white), #5 (redish), #10 (red). The white one is a good result, but the other should be white as well.

- RO1 + BB #2/3 (white)

- RO1 + BB #3/3 (white)

- RO1 + BB #1/1 (white)

- We have noticed for a few days that RO1#3 doesn't work, there might be a mutation as it never gives the right results.

- The RO1 should be red ! So it could be that the LovTAP is working (upon illumination, LovTAP binds its operator Trp and inhibits TrpO and thus RFP).

- RO2 + BB #8 (RO2 #10 + BB6, clone #8) -> slightly redish

- RO2 + BB #3 (RO2 #4 + BB1, clone #3) -> slightly redish

All have been re-inocculated (500 ul) in 25 ml of fresh M9 + antibiotic, and put in incubator at 37°C at 9.15 am.

People in the lab

Mélanie, Caroline, Basile, Nicolas