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 18th August 1.1 Lab work 1.1.1 Visualization 1.1.1.1 Fragment 3 digestion with Eco47III 1.1.1.2 Digested fragment 3 ligation with fragment 4 1.1.2 Biobrick characterization 1.1.2.1 B-Galactosidase and luciferase test on transformed BL21 Thursday 18th August Lab work Visualization Fragment 3 digestion with Eco47III By Alice gBlocks 3 were digested with eco47III enzyme following this protocol. The mix was incubated 2 hours at 37°C. Then eco47III was inactivated during 20 min at 60°C. Then digestion products were purified following this protocol. Digested fragment 3 ligation with fragment 4 By Alice Fragment 3 digested with eco47III was ligated with fragment 4. 3µL of fragment 4 amplified by PCR, 12µL of fragment 3 amplified by PCR and then digested with eco47III, 2µL of ligase buffer, 1µL of ligase and 2µL of sterile water were mix in a tube. The mix was incubated 1 hour at room temperature. Biobrick characterization B-Galactosidase and luciferase test on transformed BL21 By Charlene and Mathilde Cultures were spun down for 5min at 13000rpm. Supernatant was discarded and glass marbles previously cleaned with 1M nitric acid were added with 50μL of Luc buffer. Tubes were vortexed for 30min at 4°C. Everything was then done on ice. 150μL of Luc bufer was added and cells were centrifuged for 15min at 13000rpm at 4°C. For the luciferase test, 4mL of Luc buffer were mixed to 80μL of ATP and 8μL of luciferine. For the βGal test, 400μL of Z buffer mixed with 10μL of extract and 100μL of ONPG were incubated for 8min at 30°C. Then 250μL of STOP buffer was added. OD420nm was measured.
By Alice
gBlocks 3 were digested with eco47III enzyme following this protocol. The mix was incubated 2 hours at 37°C. Then eco47III was inactivated during 20 min at 60°C. Then digestion products were purified following this protocol.
Fragment 3 digested with eco47III was ligated with fragment 4. 3µL of fragment 4 amplified by PCR, 12µL of fragment 3 amplified by PCR and then digested with eco47III, 2µL of ligase buffer, 1µL of ligase and 2µL of sterile water were mix in a tube. The mix was incubated 1 hour at room temperature.
By Charlene and Mathilde
Cultures were spun down for 5min at 13000rpm. Supernatant was discarded and glass marbles previously cleaned with 1M nitric acid were added with 50μL of Luc buffer. Tubes were vortexed for 30min at 4°C. Everything was then done on ice. 150μL of Luc bufer was added and cells were centrifuged for 15min at 13000rpm at 4°C.
For the luciferase test, 4mL of Luc buffer were mixed to 80μL of ATP and 8μL of luciferine. For the βGal test, 400μL of Z buffer mixed with 10μL of extract and 100μL of ONPG were incubated for 8min at 30°C. Then 250μL of STOP buffer was added. OD420nm was measured.
