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 Monday 25th July 1.1 Lab work 1.1.1 Visualization 1.1.1.1 Low Fidelity Dreamtaq PCR of transformed DH5α with pPS16_002 1.1.1.2 High fidelity Q5 PCR on bacteria transformed with pPS16_004, pPS16_007 and pPS16_008 1.1.1.3 High fidelity PCR on bacteria transformed with pPS16_001, pPS16_005 and pPS16_009 1.1.2 Characterization 1.1.2.1 Culture of BL21 electrocompetent cells Monday 25th July Lab work Visualization Low Fidelity Dreamtaq PCR of transformed DH5α with pPS16_002 By Mathilde and Caroline A DreamTaq PCR was made with the transformed culture pPS16_002 re-plated the day before. The usual protocol was followeed with Tm at 57°c and 5min for the initial denaturation. We divided up the PCR mix in 6 PCR tubes and added in each one a different clone from the transformed culture. The picked colonies were re-plated on a petri dish medium LB + Ampicillin (50µg/mL) + xGal/IPTG (1/1000). Results : PCR products expected were : Plasmid pPS16_002 Band Size (bp) 960 The protocol was made again with 6 other white colonies from de same Petri dish. High fidelity Q5 PCR on bacteria transformed with pPS16_004, pPS16_007 and pPS16_008 By Caroline High fidelity Q5 PCR was carried out on colonies transformed with pPS16_004, pPS16_007 and pPS16_008 following the usual protocol adapted for 50µL final volume with puc19 universal primers. This was made in order to have more PCR products for pPS16_004 and pPS16_007 to send to sequencing. For pPS16_008 we had to send it again using PCR from new transformed colonies because we did not found the previous one. High fidelity PCR on bacteria transformed with pPS16_001, pPS16_005 and pPS16_009 By Alice Plasmids pPS16_001, pPS16_005 and pPS16_009 containing gBlocks 1.1, 3.1 and GFP 1-9 respectively, were sent to sequencing. Sequencing revealed that clones 2 transformed with pPS16_001, clone 1 transformed with pPS16_005, and clone 1 transformed with pPS16_009 had the good insert in their plasmid. In ordered to assembled gBlocks, a PCR with Q5® High-Fidelity 2X Master Mix was performed on these clones following this protocol. Specific primers were used for each plasmids (table below). Annealing temperature calculated was 72°C, and initial step was prolonged to 7 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 25min at 100V. PCR products expected were : Plasmids gBlocks Primer Forward Primer Reverse Band size (bp) pPS16_001 1.1 iPS138 iPS120 960 pPS16_005 3.1 iPS138 iPS126 960 pPS16_009 GFP 1-9 iPS138 iPS139 862 Characterization Culture of BL21 electrocompetent cells By Léa A BL21 colony was put into 4mL of liquid LB medium, and grown at 37°C, 180 rpm overnight.
By Mathilde and Caroline
A DreamTaq PCR was made with the transformed culture pPS16_002 re-plated the day before. The usual protocol was followeed with Tm at 57°c and 5min for the initial denaturation.
We divided up the PCR mix in 6 PCR tubes and added in each one a different clone from the transformed culture. The picked colonies were re-plated on a petri dish medium LB + Ampicillin (50µg/mL) + xGal/IPTG (1/1000).
Results : PCR products expected were :
The protocol was made again with 6 other white colonies from de same Petri dish.
By Caroline
High fidelity Q5 PCR was carried out on colonies transformed with pPS16_004, pPS16_007 and pPS16_008 following the usual protocol adapted for 50µL final volume with puc19 universal primers. This was made in order to have more PCR products for pPS16_004 and pPS16_007 to send to sequencing. For pPS16_008 we had to send it again using PCR from new transformed colonies because we did not found the previous one.
By Alice
Plasmids pPS16_001, pPS16_005 and pPS16_009 containing gBlocks 1.1, 3.1 and GFP 1-9 respectively, were sent to sequencing. Sequencing revealed that clones 2 transformed with pPS16_001, clone 1 transformed with pPS16_005, and clone 1 transformed with pPS16_009 had the good insert in their plasmid. In ordered to assembled gBlocks, a PCR with Q5® High-Fidelity 2X Master Mix was performed on these clones following this protocol. Specific primers were used for each plasmids (table below). Annealing temperature calculated was 72°C, and initial step was prolonged to 7 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 25min at 100V.
PCR products expected were :
By Léa
A BL21 colony was put into 4mL of liquid LB medium, and grown at 37°C, 180 rpm overnight.
