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 Just after putting the DH5αlpPS16_008 clone in the PCR tube We divided a Petri dish in 7(for 7 clones from DH5αlpPS16_008). Just after putting the DH5αlpPS16_008 clone in the PCR tube For each clone:
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:
Just after putting the DH5αlpPS16_008 clone in the PCR tube We divided a Petri dish in 7(for 7 clones from DH5αlpPS16_008). Just after putting the DH5αlpPS16_008 clone in the PCR tube
For each clone:
