Difference between revisions of "Team:Paris Saclay/Notebook/October/19"
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| + | ===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. | ||
{{Team:Paris_Saclay/notebook_footer}} | {{Team:Paris_Saclay/notebook_footer}} | ||
Revision as of 22:36, 19 October 2016
