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 Friday 8th July 1.1 Lab work 1.1.1 Visualization 1.1.1.1 Colony screening PCR on bacteria transformed with pPS16_004 and pPS16_007 1.1.1.2 Electrophoresis of the screening PCR products 1.1.1.3 High fidelity PCR Friday 8th July Lab work Visualization Colony screening PCR on bacteria transformed with pPS16_004 and pPS16_007 By Alice After transformation, only white bacteria are selected (blue white screen). 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, 5 µL of each PCR products and 10 µL of DNA ladder were placed in wells and migrated at 100v during 30 min. PCR products expected were : Plasmids expected band size (bp) pPS16_004 865 pPS16_007 763 No PCR products are observed, due to an error in the protocol : MilliQ water was used instead of steril water. The same PCR will be performed on 11/07/16. Electrophoresis of the screening PCR products By Caroline Amplification products from the 07/07/2016 screening PCR were put in a 0.8% agarose gel containing BET. The products in solution were mixed with purple loading dye and migrated for 30min. Some clones showed PCR products with lengths close to those expected. High fidelity PCR By Caroline The clones (from 05/07/2016) found with a rightful product lengths were used in another PCR this time with a high fidelity enzyme Q5. We used a high fidelity enzyme because to reduce the error ratio and sequence the PCR products. A PCR was carry out using Q5® High-Fidelity 2X Master Mix and following the usual protocol with universal pUC19 primers 1151_pheoR and 1152_pheoF. Plasmids Number of clones tested Expected band size (bp) pPS16_001 2 998 pPS16_002 3 998 pPS16_003 2 1061 pPS16_005 2 998 pPS16_006 3 998 pPS16_007 1 746
By Alice
After transformation, only white bacteria are selected (blue white screen). 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, 5 µL of each PCR products and 10 µL of DNA ladder were placed in wells and migrated at 100v during 30 min.
PCR products expected were :
No PCR products are observed, due to an error in the protocol : MilliQ water was used instead of steril water. The same PCR will be performed on 11/07/16.
By Caroline
Amplification products from the 07/07/2016 screening PCR were put in a 0.8% agarose gel containing BET. The products in solution were mixed with purple loading dye and migrated for 30min. Some clones showed PCR products with lengths close to those expected.
The clones (from 05/07/2016) found with a rightful product lengths were used in another PCR this time with a high fidelity enzyme Q5. We used a high fidelity enzyme because to reduce the error ratio and sequence the PCR products. A PCR was carry out using Q5® High-Fidelity 2X Master Mix and following the usual protocol with universal pUC19 primers 1151_pheoR and 1152_pheoF.
