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.