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 Wednesday 19th October Visualization Cytometry measurements= By Sylvie No clone was observed on the control plate from 18/10 Clones have grown on the other plates. Transformations were done from the Petri dish containing the preparation diluted 10 times with : 50 mL LB (CM 15μg/L+Amp 50μg/L) 500μL of cell preparation at DO=2,2 This preparation was divided in 4 tubes of 4ml and a falcon of 32 ml. In 1 pool of 4 tubes we added rapalog in order to test the fluorescence : tube1 : control without rapalog tube 2 : 5 nM of rapalog tube 3 : 50 nM of rapalog tube4 : 500 nM of rrapalog After 5 hours of culture, cytometry didn't show any fluorescence. For the falcon of the remaining preparation (32ml) : Centrifuge Discard the supernatant Resuspend the pellet in 15 ml of 100mM Tris (pH= 7,4), 100mM NaCl, 10 % glycerol Discardthe supernatant Resuspend in 800 μL of TNG buffer and 20μL of PMSI Divide the preparation in 8 eppendorf (100μL) For one eppendorf : add « cap of glass bead, acid washed) 150-212μm sigma » 30 min on vortex Add 100μL of buffer in each tube centrifuge Add Rapalog at 150nM in each tube except for one control Incubate 30 min Read on a board (plaque) with a 488 nm excitation.
By Sylvie
No clone was observed on the control plate from 18/10 Clones have grown on the other plates.
Transformations were done from the Petri dish containing the preparation diluted 10 times with :
50 mL LB (CM 15μg/L+Amp 50μg/L) 500μL of cell preparation at DO=2,2
This preparation was divided in 4 tubes of 4ml and a falcon of 32 ml.
In 1 pool of 4 tubes we added rapalog in order to test the fluorescence : tube1 : control without rapalog tube 2 : 5 nM of rapalog tube 3 : 50 nM of rapalog tube4 : 500 nM of rrapalog
After 5 hours of culture, cytometry didn't show any fluorescence.
For the falcon of the remaining preparation (32ml) :
Read on a board (plaque) with a 488 nm excitation.
