Toggle navigation Home Team Team Media Collaborations Sponsors Acknowledgements Project Background Design CRISPR/Cas9 Strategy Experiments Notebook Results Perspective Interlab Study Parts Parts Basic Parts Composite Parts Human Pratices Overview Societal Issues of CRISPR/Cas9 Responsible Research and Innovation GMO regulation Integrated Practices Engagement Model Attributions Safety Contents 1 Friday 24th June 1.1 Lab work 1.1.1 Heat-shock competent cells 1.1.2 Interlab study 1.1.2.1 Device 1 transformation 1.1.2.2 Plasmid DNA extraction from our transformed bacteria (cf. protocole) Friday 24th June Lab work Heat-shock competent cells By Caroline, Lea and Marion Colonies were observed with the new competent cells : the cells are competent. 41 colonies growned 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 provided by the lab. Interlab study Device 1 transformation By Caroline, Lea and Marion No colonies were observed : there was probably no DNA in the tube. Plasmid DNA extraction from our transformed bacteria (cf. protocole) By Caroline and Lea 1mL of each culture 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.
By Caroline, Lea and Marion
Colonies were observed with the new competent cells : the cells are competent. 41 colonies growned 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 provided by the lab.
No colonies were observed : there was probably no DNA in the tube.
By Caroline and Lea
1mL of each culture 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.
