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 Thursday 30th June 1.1 Lab work 1.1.1 Visualization 1.1.1.1 Extraction of the plasmids containing gBlocks 1.1.1.2 Digestion of the plasmids containing gBlocks 1.1.2 Biobrick characterization 1.1.2.1 Electrocompetents and transformation by electroporation of BL21 Thursday 30th June Lab work Visualization Extraction of the plasmids containing gBlocks By Alice and Lea DNA from bacteria transformed with plasmids containing gBlocks was extracted following the extraction protocol. For each gblock, DNA of 6 different clones was extracted. At the end, plamids were resuspended in water/RNAse (5µL RNAse 10 mg/mL for 1 mL of water) to send to sequencing. Digestion of the plasmids containing gBlocks By Alice and Léa After extraction, plasmids were digested with EcoRI and HindIII. Component Volume (µL) Plasmids 3 Red buffer 10x 2 Water 14 EcoRI enzyme 0.5 HindIII enzyme 0.5 The mix was incubated for 1 hour at 37°C and then stored at -20°C until the gel electrophoresis was done. Fragments are expected to be as follow: Plasmid name Plasmid size (kb) Digestion product size (kb) 1.1 3.7 2.7 and 1 1.2 3.7 2.7 and 1 2.1 3.7 2.7 and 1 3.1 3.7 2.7 and 1 3.2 3.7 2.7 and 1 4.1 3.4 2.7 and 0.7 4.2 4 2.7 and 1.3 GFP 1-9 3.6 2.7 and 0.9 Biobrick characterization Electrocompetents and transformation by electroporation of BL21 By Caroline and Charlene The same protocol as 29/06/16 was used for the plasmids K1372001, K1372001+pcl_TAA and the controls which were done following the same protocol but without plasmids. K1372001 and the controls were streaked on LB + Cm (30µg/mL). K137200 + PCLTAA was streaked on LB + Cm (30µg/mL) + Streptomicin (50µg/mL). This time two different streaks were made for each plasmid. The first one using 50µL from the culture. The second one was done using 100µL of each remaining culture (400µL) after concentration by centrifugation. The petri dishes made on 29/06 were stored at 4ºC
By Alice and Lea
DNA from bacteria transformed with plasmids containing gBlocks was extracted following the extraction protocol. For each gblock, DNA of 6 different clones was extracted. At the end, plamids were resuspended in water/RNAse (5µL RNAse 10 mg/mL for 1 mL of water) to send to sequencing.
By Alice and Léa
After extraction, plasmids were digested with EcoRI and HindIII.
The mix was incubated for 1 hour at 37°C and then stored at -20°C until the gel electrophoresis was done. Fragments are expected to be as follow:
By Caroline and Charlene
The same protocol as 29/06/16 was used for the plasmids K1372001, K1372001+pcl_TAA and the controls which were done following the same protocol but without plasmids. K1372001 and the controls were streaked on LB + Cm (30µg/mL). K137200 + PCLTAA was streaked on LB + Cm (30µg/mL) + Streptomicin (50µg/mL). This time two different streaks were made for each plasmid. The first one using 50µL from the culture. The second one was done using 100µL of each remaining culture (400µL) after concentration by centrifugation.
The petri dishes made on 29/06 were stored at 4ºC
