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 19 th October Cytometry measurements By Sylvie There was no clone on the control plate from 18/10. Clones have grown on the other plates. Transformations were done from the plates 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 tube of 32 ml. In the 4 tubes, we added rapalog to test the fluorescence: tube 1: control without rapalog tube 2: 5 nM of rapalog tube 3: 50 nM of rapalog tube 4: 500 nM of rapalog After 5 hours of culture, cytometry didn't show any fluorescence. For the falcon of the remaining preparation (32ml): Centrifuge Discard the supernatant Re-suspend the pellet in 15 ml of 100mM Tris (pH= 7,4), 100mM NaCl, 10 % glycerol Discard the supernatant Re-suspend in 800 μL of TNG buffer and 20μL of PMSI Divide the preparation in 8 Eppendorf (100μL) For one Eppendorf: add glass bead, acid washed (150-212μm) 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 plate at 488 nm excitation.
By Sylvie
There was no clone on the control plate from 18/10. Clones have grown on the other plates.
Transformations were done from the plates 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 tube of 32 ml.
In the 4 tubes, we added rapalog to test the fluorescence:
After 5 hours of culture, cytometry didn't show any fluorescence.
For the falcon of the remaining preparation (32ml):
Read on a plate at 488 nm excitation.
