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 Tuesday 16th August 1.1 Lab work 1.1.1 Visualization 1.1.1.1 2.1-2.2 and 3.1-3.2 ligation 1.1.1.2 Q5 PCR on the ligation products and pPS16_008 clones 1 and 2 1.1.1.3 PCR Clean-up with the NucleoSpin kit 1.1.1.4 4.1-4.2 ligation Tuesday 16th August Lab work Visualization 2.1-2.2 and 3.1-3.2 ligation By Charlène 8µL of 2.1 purify PCR products, 8µL of 2.2 purify PCR products, 2µL of ligase T4 Buffer and 2L of ligase T4 were mixed. 8µL of 3.1 purified PCR products, 8µL of 3.2 purified PCR products, 2µL of ligase T4 Buffer and 2µL of ligase T4 were mixed. They were incubated for 1h at RT. Q5 PCR on the ligation products and pPS16_008 clones 1 and 2 By Charlène The PCR was carried out with a new protocol: Q5 PCR recipe Buffer Q5 HF (5X) 10µL dNTP (10mM) 1µL Primers (10 µM, each) 2,5µL DNA 1µL for plasmid, 2µL for ligation's products Q5 DNA polymerase 0.25µL Nuclease-free water up to 50µL Steps for PCR : Step Temperature Time Initial denaturation 98°C 30sec 30 cycles 98°C 5sec Tannealing 30sec 72°C 30sec/kb Final Extension 72°C 2min Hold 4°C $\infty$ An specific primers for each part was used. The products were migrated on a 0.8% agarose gel with BET. Gel Electrophoresis of PCR products fragments 2, 3 and gblock 4.1.JPG All the bands have the good size so purifications will be done on PCR products. PCR Clean-up with the NucleoSpin kit By Mahnaz The purification was carried out on PCR product PSB1C3 (linearized) and 4.2 also the ligation products PS16003-PS16004 and PS16005-PS16006 following the usual protocol. Result of the PCR 4.1-4.2 ligation By Mahnaz 8 µL of 4.1 purified PCR product, 8µL of 4.2 purified PCR product, 2 µL of ligase T4 Buffer and 2 µL of ligase T4 were mixed. The ligation reaction mixture were incubated overnight at 4°C.
By Charlène
8µL of 2.1 purify PCR products, 8µL of 2.2 purify PCR products, 2µL of ligase T4 Buffer and 2L of ligase T4 were mixed. 8µL of 3.1 purified PCR products, 8µL of 3.2 purified PCR products, 2µL of ligase T4 Buffer and 2µL of ligase T4 were mixed. They were incubated for 1h at RT.
The PCR was carried out with a new protocol: Q5 PCR recipe
Steps for PCR :
An specific primers for each part was used.
The products were migrated on a 0.8% agarose gel with BET.
All the bands have the good size so purifications will be done on PCR products.
By Mahnaz
The purification was carried out on PCR product PSB1C3 (linearized) and 4.2 also the ligation products PS16003-PS16004 and PS16005-PS16006 following the usual protocol.
8 µL of 4.1 purified PCR product, 8µL of 4.2 purified PCR product, 2 µL of ligase T4 Buffer and 2 µL of ligase T4 were mixed. The ligation reaction mixture were incubated overnight at 4°C.
