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 29th July 1.1 Lab work 1.1.1 Visualization 1.1.1.1 gBlock 1.1, 1.2 and GFP1-9 insertion in puc19 1.1.1.2 High fidelity PCR on bacteria transformed with pPS16_001, pPS16_002 and pPS16_005 Friday 29th July Lab work Visualization gBlock 1.1, 1.2 and GFP1-9 insertion in puc19 By Caroline The insertion was carried out following the usual protocol. High fidelity PCR on bacteria transformed with pPS16_001, pPS16_002 and pPS16_005 By Alice and Mathilde A PCR with Q5® High-Fidelity 2X Master Mix was performed on clones selected after screening PCR made on July 28, following this protocol. We choose clones which had the good size of insert. We selected the clone 2 for DH5α|pPS16_001; clones 4,7 and 8 for DH5α|pPS16_002 and clones 1 and 4 DH5α|pPS16_005. 1151_pheoR and 1152_pheoF primers were used. Annealing temperature was 66°C and initial denaturation was 3 min. After amplification, 1 µL of loading dye was added to 5µL of each PCR products. Then 5 µL of this mix were put into a well of the gel. A last well contained 10µL of DNA ladder. PCR products were migrated 30min at 100V. PCR products expected were : Plasmids Band size (bp) pPS16_001 1017 pPS16_002 1017 pPS16_005 1017 File:T--Paris Saclay--160729 Visualisation PCR.TIF Migration of gBlocks PCR product
By Caroline
The insertion was carried out following the usual protocol.
By Alice and Mathilde
A PCR with Q5® High-Fidelity 2X Master Mix was performed on clones selected after screening PCR made on July 28, following this protocol. We choose clones which had the good size of insert. We selected the clone 2 for DH5α|pPS16_001; clones 4,7 and 8 for DH5α|pPS16_002 and clones 1 and 4 DH5α|pPS16_005. 1151_pheoR and 1152_pheoF primers were used. Annealing temperature was 66°C and initial denaturation was 3 min. After amplification, 1 µL of loading dye was added to 5µL of each PCR products. Then 5 µL of this mix were put into a well of the gel. A last well contained 10µL of DNA ladder. PCR products were migrated 30min at 100V.
PCR products expected were :
