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 18th July 1.1 Lab work 1.1.1 Preparation of LB solid and liquid stock 1.1.2 Getting DNA closer 1.1.2.1 Electrophoresis of PCR products DS-SPcasN-, DS-TDcasN-, pZA21 and pZA31 1.1.3 Biobrick characterization 1.1.3.1 LB culture of electro-transformed BL21 cells (cf. protocol) 1.1.4 Visualization 1.1.4.1 Electrophoresis of PCR products pPS16_004 1.1.4.2 Transformation of bacteria with pPS16_004 and pPS16_007 Monday 18th July Lab work Preparation of LB solid and liquid stock By Caroline and Terrence - 1L of LB liquid : 20g/L powder LB + 1L water μQ - 500mL of LB solid : 500mL of LB liquid + 7.5g/L of Agar The all was put in the autoclave for sterilization. Getting DNA closer Electrophoresis of PCR products DS-SPcasN-, DS-TDcasN-, pZA21 and pZA31 By Caroline PCR products obtained the 13/07/2016 were put to migrate for 30min in a 0.8% agarose gel. Migration of SP,TD, pZA21 and pZA31 Biobrick characterization LB culture of electro-transformed BL21 cells (cf. protocol) By Mathilde Each transformation : BL21|pcl_TAA + K1372001, BL21|pcl_TAG + K1372001 and BL21|pcl_Tq + K1372001 was added to 3mL of LB and one or two antibiotics. Three conditions were tested for each transformation : 15µL of Chloramphenicol (30µg/mL) 5 µL of Streptomycin (50µg/mL) 15µL of Chloramphenicol (30µg/mL) + 5 µL of Streptomycin (50µg/mL) Cultures were put in incubation at 37°C, 180 rpm. Visualization Electrophoresis of PCR products pPS16_004 By Caroline PCR products obtained the 13/07/2016 were put to migrate for 30min in a 0,8% agarose gel Transformation of bacteria with pPS16_004 and pPS16_007 By Alice, Terrence and Mathilde PCR made on bacteria transformed with pPS16_004 and pPS16_007 did not give expected results. We supposed that bacteria used for these PCRs did not still have the plasmid of interest. That is why we performed another transformation of DH5α competent cells with pPS16_004 and pPS16_007. We transformed cells following this protocol. We add 5μl of sterile H2O to the ligation product rest and used the whole for the transformation. Finally, we spread cells on Petri dishes (LB + Amp 50μg/mL + xGal 0.25μL/mL + IPTG 0.1 μL/mL) in duplicate .
By Caroline and Terrence
- 1L of LB liquid : 20g/L powder LB + 1L water μQ - 500mL of LB solid : 500mL of LB liquid + 7.5g/L of Agar The all was put in the autoclave for sterilization.
By Caroline
PCR products obtained the 13/07/2016 were put to migrate for 30min in a 0.8% agarose gel.
By Mathilde
Each transformation : BL21|pcl_TAA + K1372001, BL21|pcl_TAG + K1372001 and BL21|pcl_Tq + K1372001 was added to 3mL of LB and one or two antibiotics.
Three conditions were tested for each transformation :
Cultures were put in incubation at 37°C, 180 rpm.
PCR products obtained the 13/07/2016 were put to migrate for 30min in a 0,8% agarose gel
By Alice, Terrence and Mathilde
PCR made on bacteria transformed with pPS16_004 and pPS16_007 did not give expected results. We supposed that bacteria used for these PCRs did not still have the plasmid of interest. That is why we performed another transformation of DH5α competent cells with pPS16_004 and pPS16_007. We transformed cells following this protocol. We add 5μl of sterile H2O to the ligation product rest and used the whole for the transformation. Finally, we spread cells on Petri dishes (LB + Amp 50μg/mL + xGal 0.25μL/mL + IPTG 0.1 μL/mL) in duplicate .
