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 Visualization 1.1.1 Extraction of the plasmids containing gBlocks 1.1.2 Digestion of the plasmids containing gBlocks 1.2 Biobrick characterization 1.2.1 BL21 electrocompetent cells preparation and transformation Thursday 30th June Visualization Extraction of the plasmids containing gBlocks By Alice and Lea Plasmids pPS16_001-009 (except pPS16_004) were extracted following the extraction protocol. Each plasmids were extracted from 6 different clones of bacteria. At the end, plasmids were resuspended in water/RNAse (5µL RNAse 10 mg/mL for 1 mL of water). 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) pPS16_001 3.7 2.7 and 1 pPS16_002 3.7 2.7 and 1 pPS16_003 3.7 2.7 and 1 pPS16_005 3.7 2.7 and 1 pPS16_006 3.7 2.7 and 1 pPS16_007 3.4 2.7 and 0.7 pPS16_008 4 2.7 and 1.3 pPS16_009 3.6 2.7 and 0.9 Biobrick characterization BL21 electrocompetent cells preparation and transformation By Caroline and Charlene The usual protocol was used for transforming cells with 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). K1372001+pcl_TAA 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 with 400µL concentrated by centrifugation. The Petri dishes made on 29/06 were stored at 4ºC.
By Alice and Lea
Plasmids pPS16_001-009 (except pPS16_004) were extracted following the extraction protocol. Each plasmids were extracted from 6 different clones of bacteria. At the end, plasmids were resuspended in water/RNAse (5µL RNAse 10 mg/mL for 1 mL of water).
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 usual protocol was used for transforming cells with 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). K1372001+pcl_TAA 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 with 400µL concentrated by centrifugation.
The Petri dishes made on 29/06 were stored at 4ºC.
