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 10th August 1.1 Lab work 1.1.1 Visualization 1.1.1.1 Colony screening PCR on bacteria transformed with pPS16_010 and pPS16_011 1.1.1.2 PCR Clean-up with the NucleoSpin kit 1.1.1.3 2.1-2.2 and 3.1-3.2 ligation 1.1.1.4 GFP and PSB1C3 digestion 1.1.1.5 Phusion PCR on the ligation products 1.1.1.6 Extraction of pPS16_014(clone 3 and 11), pPS16_002 (clone 7), pPS16_009(clone 7) and pPS16_011 (clone 3, 4, 6) 1.1.2 Bringing DNA closer 1.1.2.1 Extraction of DS-SPcasN- and DS-TDcasN- Wednesday 10th August Lab work Visualization Colony screening PCR on bacteria transformed with pPS16_010 and pPS16_011 By Alice After transformation, only white bacteria are selected (blue white screen). For bacteria transformed with pPS_011, the only clone white is chosen, and for bacteria transformed with pPS16_010, 9 white clones among others are chosen. They are expected to have plasmids with inserts of interest. PCR with DreamTaq DNA Polymerase is performed following this protocol. Clones selected for PCR are kept on LB+Amp+XGal(2µLXGal/1000µLH2O)+IPTG(2µLIPTG/1000µLH2O). Two primers (1151_pheoR and 1152_pheoF) upstream and downstream the insertion site are chosen. Annealing temperature was 53°C. After amplification, 1 µL of each PCR products and 10 µL of DNA ladder were placed in wells and migrated at 100v during 30 min. This gel is also used to migrate pUC19 plamids digested with HincII. PCR products expected were : Plasmids expected band size (bp) pUC19 digested with HincII 2696 pPS16_010 431 pPS16_011 1077 Migration PCR Clean-up with the NucleoSpin kit By Caroline The purification was carried out on PCR 1.1, 3.1, 3.2, 4.1 and GFP following the usual protocol. They were put to migrated on 0.8% agarose gel containing BET. 2.1-2.2 and 3.1-3.2 ligation By Charlène 4µL of 2.1 purify PCR products, 4µL of 2.2 purify PCR products, 1µL of ligase T4 Buffer and 1µL of ligase T4 were mixed and incubated for 1h at RT. GFP and PSB1C3 digestion By Charlène 8µL of GFP purify PCR products or 8µL of PSB1C3, 1µL of Buffer, 0.5µL of EcoRI and 0.5µL of PstI were mixed and incubated for 1h at 37°C. Phusion PCR on the ligation products By Caroline The PCR was carried out following the usual protocol adapted to 50µL and with a TM at 72°C. The specific primers for each parts were using. The products were put to migrated on a 0.8%agarose gel with BET. Extraction of pPS16_014(clone 3 and 11), pPS16_002 (clone 7), pPS16_009(clone 7) and pPS16_011 (clone 3, 4, 6) By Terrence The extraction was carried out following the usual protocol. Bringing DNA closer Extraction of DS-SPcasN- and DS-TDcasN- By Terrence The extraction was carried out with the Nucleobond Ax Kit.
By Alice
PCR products expected were :
By Caroline
The purification was carried out on PCR 1.1, 3.1, 3.2, 4.1 and GFP following the usual protocol. They were put to migrated on 0.8% agarose gel containing BET.
By Charlène
4µL of 2.1 purify PCR products, 4µL of 2.2 purify PCR products, 1µL of ligase T4 Buffer and 1µL of ligase T4 were mixed and incubated for 1h at RT.
8µL of GFP purify PCR products or 8µL of PSB1C3, 1µL of Buffer, 0.5µL of EcoRI and 0.5µL of PstI were mixed and incubated for 1h at 37°C.
The PCR was carried out following the usual protocol adapted to 50µL and with a TM at 72°C. The specific primers for each parts were using. The products were put to migrated on a 0.8%agarose gel with BET.
By Terrence
The extraction was carried out following the usual protocol.
The extraction was carried out with the Nucleobond Ax Kit.
