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 11th July 1.1 Lab work 1.1.1 Biobrick characterization 1.1.1.1 Electrocompetents and transformation by electroporation of BL21 1.1.2 Bringing DNA closer 1.1.2.1 DH5α transformation with DS-NMcas, DS-SPcasN-, DS-ST1casN- and DS-TDcasN- 1.1.3 Visualization 1.1.3.1 Colony screening PCR on bacteria transformed with pPS16_004 and pPS16_007 1.1.3.2 Electrophoresis of high fidelity PCR products 1.1.3.3 High fidelity PCR Monday 11th July Lab work Biobrick characterization Electrocompetents and transformation by electroporation of BL21 By Charlène BL21 cells were grown up to OD600nm=7.14. They were then made competent and transformed with K1372001 extracted from clone 2 and pcl_TAA, pcl_TAG or pcl_Tq to create the following strains: BL21|K1372001+pcl_TAA BL21|K1372001+pcl_TAG BL21|K1372001+pcl_Tq Bringing DNA closer DH5α transformation with DS-NMcas, DS-SPcasN-, DS-ST1casN- and DS-TDcasN- By Laetitia and Mathilde DS-NMcas, DS-SPcasN-, DS-ST1casN- and DS-TDcasN- plasmids were resuspended with 5μL of sterilized water. 5 tubes were prepared : one control tube with only 50μL of DH5α heat-shock competent cells four tubes with 50μL of DH5α heat-shock competent cells + 1μL of plasmid The regular heat-shock competent cells transformation protocol was used. Transformed cells were plated in duplicata (10μL and 50μL) on 8 Petri dishes (spectinomycin 50μg/mL). Control cells were plated on another plate (spectinomycin 50μg/mL). Visualization Colony screening PCR on bacteria transformed with pPS16_004 and pPS16_007 By Alice The colony screening PCR of the 08/07/16 is performed again. After transformation, only white bacteria are selected (blue white screen). They are expected to have plasmids with inserts of interest. PCR with DreamTaq DNA Polymerase is performed following this protocol. Clones selected for PCR are kept on LB+Amp+XGal(2µLXGal/1000µLH2O)+IPTG(2µLIPTG/1000µLH2O). Two primers (1152_pheoF and 1151 5_pheoR) upstream and downstream the insertion site are chosen. PCR products expected below : Plasmids Band size (bp) pPS16_004 846 pPS16_007 744 Electrophoresis of high fidelity PCR products By Caroline The PCR products from 08/07/2016 on clones pPS16_001, pPS16_002, pPS16_003, pPS16_005,pPS16_006 and pPS16_007 were migrated into a 0.8% agarose gel containing BET for 30min. We obtained the same bands without offtarget but we can not send them to sequence because we do not have enough solution and concentration. High fidelity PCR By Caroline The same PCR as 08/07/2016 was carry out but this time adapted to get 50µL final volume. Clones from transformations with two other plasmids were tested as well: pPS16_008 and pPS16_009. Plasmids Number of clones tested Expected band size (bp) pPS16_001 2 998 pPS16_002 3 998 pPS16_003 2 1061 pPS16_005 2 998 pPS16_006 3 998 pPS16_007 1 746 pPS16_008 3 1326 pPS16_009 2 900
By Charlène
BL21 cells were grown up to OD600nm=7.14. They were then made competent and transformed with K1372001 extracted from clone 2 and pcl_TAA, pcl_TAG or pcl_Tq to create the following strains:
By Laetitia and Mathilde
DS-NMcas, DS-SPcasN-, DS-ST1casN- and DS-TDcasN- plasmids were resuspended with 5μL of sterilized water. 5 tubes were prepared :
The regular heat-shock competent cells transformation protocol was used.
Transformed cells were plated in duplicata (10μL and 50μL) on 8 Petri dishes (spectinomycin 50μg/mL). Control cells were plated on another plate (spectinomycin 50μg/mL).
By Alice
The colony screening PCR of the 08/07/16 is performed again. After transformation, only white bacteria are selected (blue white screen). They are expected to have plasmids with inserts of interest. PCR with DreamTaq DNA Polymerase is performed following this protocol. Clones selected for PCR are kept on LB+Amp+XGal(2µLXGal/1000µLH2O)+IPTG(2µLIPTG/1000µLH2O). Two primers (1152_pheoF and 1151 5_pheoR) upstream and downstream the insertion site are chosen. PCR products expected below :
By Caroline
The PCR products from 08/07/2016 on clones pPS16_001, pPS16_002, pPS16_003, pPS16_005,pPS16_006 and pPS16_007 were migrated into a 0.8% agarose gel containing BET for 30min.
We obtained the same bands without offtarget but we can not send them to sequence because we do not have enough solution and concentration.
The same PCR as 08/07/2016 was carry out but this time adapted to get 50µL final volume. Clones from transformations with two other plasmids were tested as well: pPS16_008 and pPS16_009.
