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) 1.1.4 Visualization 1.1.4.1 Electrophoresis of PCR products pPS16_004 1.1.4.2 Transformation of DH5α with 4.1 and 2.2 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. Biobrick characterization LB culture of electro-transformed BL21 cells (Cf) By Mathilde Each transformation : pclTAA + K1372001, pcl_TAG + K1372001 and 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 DH5α with 4.1 and 2.2 By Alice and Terrence As the result of PCRs on clone 2.2 and 4.1 didn't gave us expected results. We supposed that bacteria used for the PCR didn't integrate the plasmids. So we made another transformation of Dh5α competent cells. We made a dilution with the rest of the ligation product with 5μl of sterile H2O and we follow the protocol of heat shock competent cells transformation. Finally, we spread cells on Petri dishes (LB + Amp 50μg/mL + xGal 0.25μL/mL + IPTG0 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 : pclTAA + K1372001, pcl_TAG + K1372001 and 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 and Terrence
As the result of PCRs on clone 2.2 and 4.1 didn't gave us expected results. We supposed that bacteria used for the PCR didn't integrate the plasmids. So we made another transformation of Dh5α competent cells.
We made a dilution with the rest of the ligation product with 5μl of sterile H2O and we follow the protocol of heat shock competent cells transformation.
Finally, we spread cells on Petri dishes (LB + Amp 50μg/mL + xGal 0.25μL/mL + IPTG0 0.1 μL/mL) in duplicate .
