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 1.1.1.2 Liquid culture of transformed NM, TD, ST1 and SP cas9 1.1.2 Biobrick characterization 1.1.2.1 Electrocompetents and transformation by electroporation of BL21 1.1.3 Visualization 1.1.3.1 Transformation of gBlock 4.2 pPS16_008 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. Liquid culture of transformed NM, TD, ST1 and SP cas9 By Mathilde and Laetitia Two colonies from each petri dishes were cultured in 1mL of LB and Spectinomycin (50µg/mL). Our 8 cultures were put in incubation at 37°c, 180 rpm OV. 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. Visualization Transformation of gBlock 4.2 pPS16_008 By Mathilde and Laetitia The gBlock 4.2 in the plasmid pPS16_008 was transformed with DH5α chimio competent cells following the protocol from the 11/07/2016. Petri dishes peparation (x3) : 10µL of Ampycillin (50µg/mL) 5µL of Xgal (0,25µL/mL) 2µL of IPTG (0,1µL/mL) 20 mL of LB+Agar The two gBlocks constructions were plated in duplicata (50µL and 150µL), and a controle construction was plated alone. The plates were put in incubation at 37°c overnight.
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 Mathilde and Laetitia
Two colonies from each petri dishes were cultured in 1mL of LB and Spectinomycin (50µg/mL). Our 8 cultures were put in incubation at 37°c, 180 rpm OV.
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.
The gBlock 4.2 in the plasmid pPS16_008 was transformed with DH5α chimio competent cells following the protocol from the 11/07/2016.
Petri dishes peparation (x3) : 10µL of Ampycillin (50µg/mL) 5µL of Xgal (0,25µL/mL) 2µL of IPTG (0,1µL/mL) 20 mL of LB+Agar
The two gBlocks constructions were plated in duplicata (50µL and 150µL), and a controle construction was plated alone. The plates were put in incubation at 37°c overnight.
