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 20th July 1.1 Lab work 1.1.1 1.1.2 Biobrick characterization 1.1.2.1 pclTAA, pclTAG and pclTq transformations 1.1.2.2 1.1.3 Visualization 1.1.3.1 High fidelity PCR on bacteria transformed with pPS16_005 1.1.3.2 Culture of bacteria transformed with pPS16_002 and pPS16_004 1.1.3.3 1.1.4 Get DNA Closer 1.1.4.1 Electrophoresis of PCR products DS_SPcasN, DS_TDcasN and pZA21 1.1.4.2 pZA21 transformation 1.1.4.3 Pre-culture of SP and TD Wednesday 20th July Lab work By Biobrick characterization pclTAA, pclTAG and pclTq transformations By Laetitia and Caroline In order to restore our plasmids stocks heatShock competent cells were transformed following the usual protocol with pclTAA, pclTAG and pclTq. A control was made with cells without plasmids. The transformed bacteria resulting will be put on glycerol. Cells were plated on LB + Streptomycin (50µg/ml) and incubated overnight at 37°C. By Visualization High fidelity PCR on bacteria transformed with pPS16_005 By Alice High fidelity PCR on bacteria transformed with pPS16_005 did not get PCR products. We supposed that there was a problem with iPS83 primer dilution. That is why we performed again this PCR after diluting again theses primers. We followed the same protocol as previously. iPS83 and iPS126 primers were used. Annealing temperature 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 Band size (bp) pPS16_005 964 We did not observed PCR products. This is maybe due to a iPS83 primer default. New primers iPS83 will be ordered to perform again this PCR. Culture of bacteria transformed with pPS16_002 and pPS16_004 By Alice After transformation made on the 19/07/16, bacteria were spread on petri dishes without working XGal and IPTG, explaining that we get white colonies only. These colonies were spread again on petri dishes with LB, Ampicilin (50µg/mL), XGal (0.25µL/mL) and IPTG (0.1µL/mL). By Get DNA Closer Electrophoresis of PCR products DS_SPcasN, DS_TDcasN and pZA21 By Caroline PCR products obtained the 19/07/2016 were put to migrate for 30min in a 0,8% agarose gel. pZA21 transformation By Laetitia and Caroline In order to restore our plasmids stocks heatShock competent cells were transformed following the usual protocol with pZA21. A control was made with cells without plasmids. The transformed bacteria resulting will be put on glycerol. Cells were plated on LB + Kanamycin (50µg/ml) and incubated overnight at 37°C. Pre-culture of SP and TD By Laetitia and Caroline For each Cas (SP and TD), we picked an unique colony from a petri dish and soaked it in medium of LB (4mL) and spectinomycin (2.6 µL. Then we incubated it at 37°C with agitation at 180 rpm overnight.
By
By Laetitia and Caroline
In order to restore our plasmids stocks heatShock competent cells were transformed following the usual protocol with pclTAA, pclTAG and pclTq. A control was made with cells without plasmids. The transformed bacteria resulting will be put on glycerol. Cells were plated on LB + Streptomycin (50µg/ml) and incubated overnight at 37°C.
By Alice
High fidelity PCR on bacteria transformed with pPS16_005 did not get PCR products. We supposed that there was a problem with iPS83 primer dilution. That is why we performed again this PCR after diluting again theses primers. We followed the same protocol as previously. iPS83 and iPS126 primers were used. Annealing temperature 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 :
We did not observed PCR products. This is maybe due to a iPS83 primer default. New primers iPS83 will be ordered to perform again this PCR.
After transformation made on the 19/07/16, bacteria were spread on petri dishes without working XGal and IPTG, explaining that we get white colonies only. These colonies were spread again on petri dishes with LB, Ampicilin (50µg/mL), XGal (0.25µL/mL) and IPTG (0.1µL/mL).
By Caroline
PCR products obtained the 19/07/2016 were put to migrate for 30min in a 0,8% agarose gel.
In order to restore our plasmids stocks heatShock competent cells were transformed following the usual protocol with pZA21. A control was made with cells without plasmids. The transformed bacteria resulting will be put on glycerol. Cells were plated on LB + Kanamycin (50µg/ml) and incubated overnight at 37°C.
For each Cas (SP and TD), we picked an unique colony from a petri dish and soaked it in medium of LB (4mL) and spectinomycin (2.6 µL. Then we incubated it at 37°C with agitation at 180 rpm overnight.
