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 Monday 12th July 1.1 Lab work 1.1.1 Getting DNA closer 1.1.1.1 Amplification of DS-TDcasN-, DS-SPcasN- and pZA21 plasmids 1.1.2 Biobrick characterization 1.1.2.1 Electrocompetents and transformation by electroporation of BL21 Monday 12th July Lab work Getting DNA closer Amplification of DS-TDcasN-, DS-SPcasN- and pZA21 plasmids By Caroline Plasmids extracted the 4/07/16 were used to amplify the dCas9 needed for the get DNA closer tool and the pZA21 plasmid in which the tool will be inserted. A PCR was carry out following PCR with Q5® High-Fidelity 2X Master Mix from usual protocol adapted to get 50µL at the end. Biobrick characterization Electrocompetents and transformation by electroporation of BL21 By Charlene Cells transformed on 11/07/16 grew overnight. However no colony was observed. To control if there is a bacteria transformed which was not put on Petri dish, we performed another experiment. 100µL (500µL concentrated) of transformed bacteria for each condition, conserved at -4°C from yesterday, were split in 3 solutions : 30µL of bacteria + 3mL LB + Streptomycin (50µg/mL) + Chloramphenicol (30µg/mL) 30µL of bacteria + 3mL LB + Streptomycin (50µg/mL) 30µL of bacteria + 3mL LB + Chloramphenicol (30µg/mL) Cells were incubated for 8h at 37°C, 200 RPM. No bacteria grew so we concluded that there was a problem with the transformation.
By Caroline
Plasmids extracted the 4/07/16 were used to amplify the dCas9 needed for the get DNA closer tool and the pZA21 plasmid in which the tool will be inserted. A PCR was carry out following PCR with Q5® High-Fidelity 2X Master Mix from usual protocol adapted to get 50µL at the end.
By Charlene
Cells transformed on 11/07/16 grew overnight. However no colony was observed. To control if there is a bacteria transformed which was not put on Petri dish, we performed another experiment. 100µL (500µL concentrated) of transformed bacteria for each condition, conserved at -4°C from yesterday, were split in 3 solutions :
Cells were incubated for 8h at 37°C, 200 RPM.
No bacteria grew so we concluded that there was a problem with the transformation.
