User:Dileep D. Monie/Notebook/2008-4-2: Difference between revisions
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*Diluted 50 μl of overnight culture into 5 ml of pre-warmed (37°C) fresh media (1:100) | *Diluted 50 μl of overnight (20 h) culture into 5 ml of pre-warmed (37°C) fresh media (1:100) | ||
*Grew for 4 h at 37°C with shaking at 225 RPM | *Grew for 4 h at 37°C with shaking at 225 RPM | ||
**Note that I '''used a higher RPM''' than mentioned in the [[The_BioBricks_Foundation:Standards/Technical/Measurement/Promoter_characterization_experiment_(FACS)|protocol]] | **Note that I '''used a higher RPM''' than mentioned in the [[The_BioBricks_Foundation:Standards/Technical/Measurement/Promoter_characterization_experiment_(FACS)|protocol]] | ||
Revision as of 11:02, 3 April 2008
- Diluted 50 μl of overnight (20 h) culture into 5 ml of pre-warmed (37°C) fresh media (1:100)
- Grew for 4 h at 37°C with shaking at 225 RPM
- Note that I used a higher RPM than mentioned in the protocol
- Observed turbidity visually after 4 h
- Pelleted 1 ml of each culture in a 1.5 ml microcentrifuge tube at 3000 RCF for 5 min at RT
- Pellet sizes were all similar except for the three I20260 cells, which appeared smaller
- Stored remaining cultures at 4°C for future OD600 readings (if necessary)
- Removed the supernatant
- Resuspended the cell pellets in 1 ml of 1X PBS
- Added 500 μl of resuspended cells through a cell strainer lid into a 5 ml polystyrene tube
- Placed cells on ice
- Analyzed cells by flow cytometry using a BD FACSCalibur withing 30 min
- Captured 250,000 events per sample (this seems to be far more than necessary)
- Observed fluorescence in the FL1 channel
- Used the following instrument settings:
