Friday 24^th June

Heat-shock competent cells

By Caroline, Lea and Marion

Colonies were observed with the new competent cells which testify that the cells are competent. 41 colonies grown in dishes which received 50µL of transformation product for the Interlab Study negative control with the new competent cells and approximately 400 colonies for the pSB1C3 blue plasmid. These results were compared with results obtained with the competent cells we used on 21/06/2016 : our competent cells were approximately as competent as competent cells previously provided by the lab.

Interlab study

Device 1 transformation

By Caroline, Lea and Marion

No colonies were observed. We suppose that there was probably no DNA in the tube.

Plasmid DNA extraction from our transformed bacteria (cf. protocol)

By Caroline and Lea

1mL of each culture tested the 23/06/2016 was put in 500µL of glycerol and frozen.

What was left of each culture was centrifuged at 13000rpm for 1min in order to pellet the cells. This pellet was resuspended with 100µL of TE. 200µL of solution II was added and bacteria were gently mixed until lysates appear clear (dissolution). Then 150µL of solution III was added and bacteria were gently mixed again to induce precipitation. The solutions were kept on ice for 10min and centrifuged 10min at 13000rpm. Supernatants were recovered. 100µL of phenol was added in each tube to denature the proteins, tubes were vortexed for 30seconds and centrifuged for 7min at 13000rpm. The aqueous phases, which contain the DNA, were recovered. 2 volumes (900µL) of 100% ethanol were added and solutions were put into -20°C for 10min. Then, the tubes were centrifuged for 10min at 13000rpm. Supernatants were discarded and 800µL of 70% ethanol was added to remove ions, making sure that the pellet containing DNA remained at the bottom of the tube. The tubes were centrifuged 4min at 13000rpm and supernatants were removed. The tubes were dried in speedvac and the pellets were resuspended in 50µL of TE/RNAse. Tubes were kept at -20°C.