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 Tuesday 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 Electrophoresis of the PCR amplification 1.1.1.3 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 Tuesday 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 using Q5® High-Fidelity 2X Master Mix and following the usual protocol adapted to have 50µL at the end. Electrophoresis of the PCR amplification By Caroline 5µL of the amplifications are mixed with 1µL of purple loading dye. The solutions are put into a 0.8% agarose gel and migrated for 30min. 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 ON. 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. 100 µL (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 Laetitia and Mathilde DH5α heat-shock competent cells were transformed with plasmid pPS16_008 (gBlock 4.2). Petri dishes peparation (x3) : 10µL of Ampicillin (50µg/mL) 5µL of Xgal (0.25µL/mL) 2µL of IPTG (0.1µL/mL) 20mL of LB+Agar Cells transformed with the two gBlocks constructions were plated in duplicata (50µL and 150µL) and a control construct 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 using Q5® High-Fidelity 2X Master Mix and following the usual protocol adapted to have 50µL at the end.
5µL of the amplifications are mixed with 1µL of purple loading dye. The solutions are put into a 0.8% agarose gel and migrated for 30min.
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 ON.
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. 100 µL (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.
By Laetitia and Mathilde
DH5α heat-shock competent cells were transformed with plasmid pPS16_008 (gBlock 4.2).
Petri dishes peparation (x3) :
Cells transformed with the two gBlocks constructions were plated in duplicata (50µL and 150µL) and a control construct was plated alone. The plates were put in incubation at 37°c overnight.
