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 Phusion PCR of ligations products 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_010 374 pPS16_011 1020 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 Phusion PCR of ligations products By Caroline
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
