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 Meeting 1.2 Lab work 1.2.1 Visualization 1.2.1.1 Low Fidelity Dreamtaq PCR of DH5α|pPS16_002 1.2.1.2 Replica plating of DH5α|pPS16_002 culture 1.2.1.3 High fidelity Q5 PCR on bacteria transformed with pPS16_004, pPS16_007 and pPS16_008 1.2.1.4 High fidelity PCR on bacteria transformed with pPS16_001, pPS16_005 and pPS16_009 1.2.2 Biobrick Characterization 1.2.2.1 Culture of BL21 electrocompetent cells Monday 25th July Lab Meeting Members present: Instructors and advisors: Claire, Fabio, Marie, Sylvie. Students: Carla, Caroline, Charlène, Claire, Coline, Léa, Marion, Marin, Laetitia. Lab work Visualization Low Fidelity Dreamtaq PCR of DH5α|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 followed 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) + X-Gal/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. The electrophoresis on agarose gel showed no PCR products. However, the culture may has stayed on incubation for too long. Thus satellites ampicillin sensitives white bacteria may have grown around blue colonies misleading us with the picking process of white clones. Replica plating of DH5α|pPS16_002 culture ‘‘By Charlène and Laetitia‘‘ In order to obtain white ampicillin resistant colonies workable on PCR, the DH5α|pPS16_002 culture was replicated on LB + Ampicillin (50µg/mL) + X-Gal/IPTG (1/1000). This replica was put in incubation ON at 37°c. 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. Results of PCR products (Gblocks 2.2, 4.1, 4.2) electrophoresis 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 GFP1-9 iPS138 iPS139 862 No PCR products were expected. We supposed that initial denaturation was too long, and caused enzyme damages. Biobrick 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.
Members present:
By Mathilde and Caroline
A DreamTaq PCR was made with the transformed culture pPS16_002 re-plated the day before. The usual protocol was followed 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) + X-Gal/IPTG (1/1000).
Results : PCR products expected were :
The protocol was made again with 6 other white colonies from de same Petri dish.
The electrophoresis on agarose gel showed no PCR products. However, the culture may has stayed on incubation for too long. Thus satellites ampicillin sensitives white bacteria may have grown around blue colonies misleading us with the picking process of white clones.
‘‘By Charlène and Laetitia‘‘
In order to obtain white ampicillin resistant colonies workable on PCR, the DH5α|pPS16_002 culture was replicated on LB + Ampicillin (50µg/mL) + X-Gal/IPTG (1/1000). This replica was put in incubation ON at 37°c.
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 :
No PCR products were expected. We supposed that initial denaturation was too long, and caused enzyme damages.
By Léa
A BL21 colony was put into 4mL of liquid LB medium, and grown at 37°C, 180 rpm overnight.
