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 10th August 1.1 Lab work 1.1.1 Visualization 1.1.1.1 Colony screening PCR on bacteria transformed with pPS16_010 and pPS16_011 1.1.1.2 PCR Clean-up with the NucleoSpin kit 1.1.1.3 2.1-2.2 and 3.1-3.2 ligation 1.1.1.4 GFP and PSB1C3 digestion 1.1.1.5 Phusion PCR on the ligation products 1.1.1.6 Extraction of pPS16_002 (clone 7), pPS16_011 (clones 3, 4, 6), pPS16_014 (clones 3 and 11) and pPS16_009(clone 7) 1.1.1.7 Transformation of ligation products and pUC 19 1.1.2 Bringing DNA closer 1.1.2.1 Extraction of DS-SPcasN- and DS-TDcasN- Wednesday 10th August Lab work Visualization Colony screening PCR on bacteria transformed with pPS16_010 and pPS16_011 By Alice After transformation, only white bacteria were selected (blue and white screen). For bacteria transformed with pPS_011, the only clone white was chosen, and for bacteria transformed with pPS16_010, 9 white clones among others were chosen. They are expected to have plasmids with the wanted inserts. 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, 1 µL of each PCR products and 10 µL of DNA ladder were placed in wells and migrated at 100v during 30 min. This gel was also used to migrate pUC19 plasmids digested with HincII. PCR products expected were : Plasmids expected band size (bp) pUC19 digested with HincII 2696 pPS16_010 431 pPS16_011 1077 Migration of pPS16_010 and pPS16_011 For pPS16_010 we expected a band at about 0.4kB, that we can not see on this first gel, that is why we migrated again PCR products from clones 3,5,7 and 8 for bacteria transformed with pPS16_010 and clone 1 for bacteria transformed with pPS16_011. Migration of pPS16_010 (extracted from clones 3,5,7 and 8) and pPS16_011 (clone 1), 20 min Migration of pPS16_010 (extracted from clones 3,5,7 and 8) and pPS16_011 (clone 1), 30 min PCR Clean-up with the NucleoSpin kit By Caroline The purification was carried out on PCR 1.1, 3.1, 3.2, 4.1 and GFP following the usual protocol. They were put to migrated on 0.8% agarose gel containing BET. 2.1-2.2 and 3.1-3.2 ligation By Charlène 4µL of 2.1 purify PCR products, 4µL of 2.2 purify PCR products, 1µL of ligase T4 Buffer and 1µL of ligase T4 were mixed. 4µL of 3.1 purify PCR products, 4µL of 3.2 purify PCR products, 1µL of ligase T4 Buffer and 1µL of ligase T4 were mixed. They were incubated for 1h at RT. GFP and PSB1C3 digestion By Charlène 8µL of GFP purify PCR products or 8µL of PSB1C3, 1µL of Buffer, 0.5µL of EcoRI and 0.5µL of PstI were mixed and incubated for 1h at 37°C. Phusion PCR on the ligation products By Caroline The PCR was carried out following the usual protocol adapted to 50µL and with a TM at 72°C. The specific primers for each parts were using. The products were put to migrated on a 0.8%agarose gel with BET. Extraction of pPS16_002 (clone 7), pPS16_011 (clones 3, 4, 6), pPS16_014 (clones 3 and 11) and pPS16_009(clone 7) By Terrence The extraction was carried out following the usual protocol. Extraction of pPS16_002 (clone 7), pPS16_011 (clones 3, 4, 6), pPS16_014 (clones 3 and 11) and pPS16_009 (clone 7) Nano Drop: Plasmid name Concentration (ng/µL) 260/230 260/280 pPS16_011 clone 3 34.08 1.54 1.57 pPS16_011 clone 4 368.42 2.36 1.92 pPS16_011 clone 6 295.99 2.29 1.91 pPS16_014 clone 11 529.17 2.39 1.91 pPS16_002 clone 7 30.50 1.76 2.13 pPS16_009 clone 7 282.1 2.31 1.93 Transformation of ligation products and pUC 19 By Laetitia Heat choc transformation was performed on DH5a with the products of ligation (containing 1.2, 4.2, ATG link FRB, ATG linf FKBP, ST sg RNA, NM sg RNA and detection) following the usual protocol. Each transformation product was plated on a meduim of LB, AMpicillin IPTG and Xgal and put at 37°C Overnight. Bringing DNA closer Extraction of DS-SPcasN- and DS-TDcasN- By Terrence The extraction was carried out with the Nucleobond Ax Kit. Extraction of DS-SPcasN- and DS-TDcasN- Nano Drop: Plasmid name Concentration (ng/µL) 260/230 260/280 DS-SPcasN- 185.46 2.23 1.79 DS-TDcasN 198.97 1.88 1.79
By Alice
After transformation, only white bacteria were selected (blue and white screen). For bacteria transformed with pPS_011, the only clone white was chosen, and for bacteria transformed with pPS16_010, 9 white clones among others were chosen. They are expected to have plasmids with the wanted inserts. 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, 1 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 :
For pPS16_010 we expected a band at about 0.4kB, that we can not see on this first gel, that is why we migrated again PCR products from clones 3,5,7 and 8 for bacteria transformed with pPS16_010 and clone 1 for bacteria transformed with pPS16_011.
By Caroline
The purification was carried out on PCR 1.1, 3.1, 3.2, 4.1 and GFP following the usual protocol. They were put to migrated on 0.8% agarose gel containing BET.
By Charlène
4µL of 2.1 purify PCR products, 4µL of 2.2 purify PCR products, 1µL of ligase T4 Buffer and 1µL of ligase T4 were mixed. 4µL of 3.1 purify PCR products, 4µL of 3.2 purify PCR products, 1µL of ligase T4 Buffer and 1µL of ligase T4 were mixed. They were incubated for 1h at RT.
8µL of GFP purify PCR products or 8µL of PSB1C3, 1µL of Buffer, 0.5µL of EcoRI and 0.5µL of PstI were mixed and incubated for 1h at 37°C.
The PCR was carried out following the usual protocol adapted to 50µL and with a TM at 72°C. The specific primers for each parts were using. The products were put to migrated on a 0.8%agarose gel with BET.
By Terrence
The extraction was carried out following the usual protocol.
Nano Drop:
By Laetitia
Heat choc transformation was performed on DH5a with the products of ligation (containing 1.2, 4.2, ATG link FRB, ATG linf FKBP, ST sg RNA, NM sg RNA and detection) following the usual protocol.
Each transformation product was plated on a meduim of LB, AMpicillin IPTG and Xgal and put at 37°C Overnight.
The extraction was carried out with the Nucleobond Ax Kit.
