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 Wednesday 29th June 1.1 Lab work 1.1.1 Visualization 1.1.1.1 Culture of clones with gBlocks 1.1.2 BioBrick characterization 1.1.2.1 BL21 electro-competent cells preparation 1.1.2.2 BL21 electro-competent cells transformation 1.1.2.3 K1372001 plasmid digestion 1.1.3 Bringing DNA closer 1.1.3.1 Plasmids digestion Wednesday 29th June Lab work Visualization Culture of clones with gBlocks By Lea and Marion 6 clones of each construction were selected and grown overnight in 3.5mL of LB (50µg/ml Amp) at 37°C, 180rpm. BioBrick characterization BL21 electro-competent cells preparation By Charlene 200µL of BL21 pre-culture was added to 15mL of LB and incubated for 4h at 37°C. When abs600nm=0.68, cells were centrifuged for 10 minutes at 4000rpm and washed twice with addition of 10mL glycerol (10%) followed by 10 minutes of centrifugation at 4000rpm. Cells were mixed with 200µL glycerol and kept -80°C. BL21 electro-competent cells transformation By Charlene We realized four transformations with the following plasmids: K1372001 K1372001+pcl_TAA K1372001+pcl_TAG K1372001+pcl_Tq 50µL of cells were added in the cuvettes with 1µL DNA (diluted 1/10). Everything was done at 4°C. Transformation did not work for K1372001 and K1372001+pcl_TAA because the cuvettes were broken. Template:Font color. Cells died during transformation. Transformation worked for K1372001+pcl_TAG and K1372001+pcl_Tq. Template:Font color. We added 1mL of LB to the cells and incubated them for 1h at 37°C. The following cells were plated on Petri dishes (30µg/mL Cm + 50µg/mL streptomycin): BL21|K1372001+pcl_TAG : 50µL of transformed cells BL21|K1372001+pcl_TAG : 500µL of transformed cells concentrated 5x. BL21|K1372001+pcl_Tq : 50µL cells BL21|K1372001+pcl Tq : 500µL of transformed cells concentrated 5x. K1372001 plasmid digestion By Naiane K1372001 plasmid was digested with BglI using the following quantities: 3 µL DNA, 2µL buffer (O), 1 µL enzyme (BglI), 14µL water. The mix was incubated for 1h at 37°C and 10 min at 55°C and kept at -20°C until the gel electrophoresis was done. Fragments are expected to size 1.2kb and 2.4kb. Bringing DNA closer Plasmids digestion By Naiane
By Lea and Marion
6 clones of each construction were selected and grown overnight in 3.5mL of LB (50µg/ml Amp) at 37°C, 180rpm.
By Charlene
200µL of BL21 pre-culture was added to 15mL of LB and incubated for 4h at 37°C. When abs600nm=0.68, cells were centrifuged for 10 minutes at 4000rpm and washed twice with addition of 10mL glycerol (10%) followed by 10 minutes of centrifugation at 4000rpm. Cells were mixed with 200µL glycerol and kept -80°C.
We realized four transformations with the following plasmids:
50µL of cells were added in the cuvettes with 1µL DNA (diluted 1/10). Everything was done at 4°C.
Transformation did not work for K1372001 and K1372001+pcl_TAA because the cuvettes were broken. Template:Font color. Cells died during transformation. Transformation worked for K1372001+pcl_TAG and K1372001+pcl_Tq. Template:Font color. We added 1mL of LB to the cells and incubated them for 1h at 37°C.
The following cells were plated on Petri dishes (30µg/mL Cm + 50µg/mL streptomycin):
By Naiane
K1372001 plasmid was digested with BglI using the following quantities: 3 µL DNA, 2µL buffer (O), 1 µL enzyme (BglI), 14µL water. The mix was incubated for 1h at 37°C and 10 min at 55°C and kept at -20°C until the gel electrophoresis was done. Fragments are expected to size 1.2kb and 2.4kb.
