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 Bringing 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 and electrophoresis 1.1.3.2 Culture of DH5αlpPS16_008 clones on petri dish 1.1.3.3 Visualization 1.1.3.4 Colony screening PCR on bacteria transformed with [[Team:Paris_Saclay/Notebook/July/4#pPS16_004|pPS16_004] Wednesday 13th July Lab work Bringing 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 and electrophoresis 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: The products from the PCR were migrated on gel by electrophoresis during 20 min. 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. Visualization Colony screening PCR on bacteria transformed with [[Team:Paris_Saclay/Notebook/July/4#pPS16_004|pPS16_004] By Alice and Caroline Clones of bacteria transformed with pPS16_004 selected on the 11/07/16 did not grow on petri dishes. That why we performed again colony screening PCR on these bacteria. After transformation, only white bacteria were selected (blue white screen). They were expected to have plasmids with inserts of interest. PCR with DreamTaq DNA Polymerase was performed following this protocol. Clones selected for PCR were kept on LB+Amp+XGal(2µLXGal/1000µLH2O)+IPTG(2µLIPTG/1000µLH2O). Two primers (1151_pheoR and 1152_pheoF) upstream and downstream the insertion site were chosen. Annealing temperature was 53°C. After amplification, 5 µL of each PCR products and 10 µL of DNA ladder were placed in wells and migrated at 100v during 30 min. PCR products expected were: Plasmids Band size (bp) pPS16_004 865
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:
The products from the PCR were migrated on gel by electrophoresis during 20 min.
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 Alice and Caroline
Clones of bacteria transformed with pPS16_004 selected on the 11/07/16 did not grow on petri dishes. That why we performed again colony screening PCR on these bacteria. After transformation, only white bacteria were selected (blue white screen). They were expected to have plasmids with inserts of interest. PCR with DreamTaq DNA Polymerase was performed following this protocol. Clones selected for PCR were kept on LB+Amp+XGal(2µLXGal/1000µLH2O)+IPTG(2µLIPTG/1000µLH2O). Two primers (1151_pheoR and 1152_pheoF) upstream and downstream the insertion site were chosen. Annealing temperature was 53°C. After amplification, 5 µL of each PCR products and 10 µL of DNA ladder were placed in wells and migrated at 100v during 30 min.
PCR products expected were:
