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 the 4 tubes we added rapalog in order 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 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 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
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 the 4 tubes we added rapalog in order 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.
