Difference between revisions of "Team:Paris Saclay/Notebook/October/19"
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===Cytometry measurements=== | ===Cytometry measurements=== | ||
''By Sylvie'' | ''By Sylvie'' | ||
| − | + | There was no clone on the control plate from 18/10. | |
Clones have grown on the other plates. | Clones have grown on the other plates. | ||
| − | Transformations were done from the | + | Transformations were done from the plates containing the preparation diluted 10 times with: |
| − | + | ||
50 mL LB (CM 15μg/L+Amp 50μg/L) | 50 mL LB (CM 15μg/L+Amp 50μg/L) | ||
500μL of cell preparation at DO=2,2 | 500μL of cell preparation at DO=2,2 | ||
| − | This preparation was divided in 4 tubes of 4ml and a falcon of 32 ml. | + | This preparation was divided in 4 tubes of 4ml and a falcon tube of 32 ml. |
| − | In the 4 tubes we added rapalog | + | In the 4 tubes, we added rapalog to test the fluorescence: |
*tube 1: control without rapalog | *tube 1: control without rapalog | ||
*tube 2: 5 nM of rapalog | *tube 2: 5 nM of rapalog | ||
| Line 27: | Line 20: | ||
| − | For the falcon of the remaining preparation (32ml) : | + | For the falcon of the remaining preparation (32ml): |
*Centrifuge | *Centrifuge | ||
*Discard the supernatant | *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 | + | *Divide the preparation in 8 Eppendorf (100μL) |
| − | *For one | + | *For one Eppendorf: add glass bead, acid washed (150-212μm) |
*30 min on vortex | *30 min on vortex | ||
*Add 100μL of buffer in each tube | *Add 100μL of buffer in each tube | ||
Revision as of 23:53, 19 October 2016
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.
