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 13th July 1.1 Lab work 1.1.1 Getting DNA closer 1.1.1.1 Glycerol stocks for DH5α tra,sformed with DS-NMcas, DS-SPcasN-, DS-ST1casN- and DS-TDcasN- 1.1.1.2 Amplification of DS_SPcasN, DS_TDcasN, pZA21 and pZA31 by Phusion PCR 1.1.2 Biobrick characterization 1.1.2.1 BL21 electrocompetent cells preparation and transformation 1.1.3 Visualization 1.1.3.1 PCR of DH5αlpPS16_008 with Dream Taq 1.1.3.2 Culture of DH5αlpPS16_008 clones on petri dish Wednesday 13th July Lab work Getting DNA closer Glycerol stocks for DH5α tra,sformed with DS-NMcas, DS-SPcasN-, DS-ST1casN- and DS-TDcasN- By Laetitia 8 stocks were made: 2 clones (Cl 1 and Cl 2) by cas: Cl1 and Cl2 of DS-NMcas Cl1 and Cl2 of DS-SPcasN- Cl1 and Cl2 of DS-ST1casN- Cl1 and Cl2 of DS-TDcasN- For 1 glycerol stock: 1mL of liquid culture 500 μL of glycerol 60% Amplification of DS_SPcasN, DS_TDcasN, pZA21 and pZA31 by Phusion PCR By Caroline The different parts needed for de getting DNA closer tool were amplify by high fidelity PCR using Phusion following usual protocol with a TM at 60°C. Biobrick characterization BL21 electrocompetent cells preparation and transformation By Mathilde and Charlène We did an electro-transformation of BL21 with : pcl_TAA + K1372001 (time constant equal to 5.9ms) pcl_TAG + K1372001 (time constant equal to 6 ms) pcl_Tq + K1372001 (time constant equal to 6 ms) Cells were displayed on LB + Streptomycin (50µg/mL) + Chloramphenicol (30µg/mL) medium. For each condition, we made a dish with 50µL of cells and another with 500µL of cells. Visualization PCR of DH5αlpPS16_008 with Dream Taq By Laetitia Preparation of the PCR mix (total of 200μL ): 20 μL of Dream Taq Green Buffer 20 μL of dNTP (10 mM) 8 μL of primer 1151 (10 mM) 8 μL of primer 1152 (10 mM) 1,04 μL of Dream Taq 142,96 μL of Nuclease free water We divided up the PCR mix in 7 PCR tubes: put 25 μL of the mix in each tube. For each tube (x7 different clones) : We picked 1 white clone in the petri dish containing DH5αlpPS16_008 (from 12/07/16) and soaked it in the tube (with PCR mix) For the primers used, Tm is 57°C PCR Program: Culture of DH5αlpPS16_008 clones on petri dish By Laetitia We divided a Petri dish in 7(for 7 clones from DH5αlpPS16_008). Just after putting the DH5αlpPS16_008 clone in the PCR tube, spread the cells with the stick on the part corresponding to the clone on the petri dish. We left it grow at room temperature.
By Laetitia
8 stocks were made: 2 clones (Cl 1 and Cl 2) by cas:
For 1 glycerol stock:
By Caroline
The different parts needed for de getting DNA closer tool were amplify by high fidelity PCR using Phusion following usual protocol with a TM at 60°C.
By Mathilde and Charlène
We did an electro-transformation of BL21 with :
Cells were displayed on LB + Streptomycin (50µg/mL) + Chloramphenicol (30µg/mL) medium. For each condition, we made a dish with 50µL of cells and another with 500µL of cells.
Preparation of the PCR mix (total of 200μL ):
We divided up the PCR mix in 7 PCR tubes: put 25 μL of the mix in each tube.
For each tube (x7 different clones) : We picked 1 white clone in the petri dish containing DH5αlpPS16_008 (from 12/07/16) and soaked it in the tube (with PCR mix)
For the primers used, Tm is 57°C
PCR Program:
We divided a Petri dish in 7(for 7 clones from DH5αlpPS16_008). Just after putting the DH5αlpPS16_008 clone in the PCR tube, spread the cells with the stick on the part corresponding to the clone on the petri dish.
We left it grow at room temperature.
