User:Dileep D. Monie/Notebook/BBF Standards Measurement/2008/04/02

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2008-04-02

  • 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 cultures, which all 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 (15 mW 488 nm, air-cooled argon-ion laser) within 30 min
    • Captured 250,000 events per sample (this seems to be far more than necessary)
    • Observed fluorescence in the FL1 channel (530/30 BP emission filter)
    • Used the following instrument settings (threshold was not optimized for samples):
Detectors/Amps:
Param  Detector  Voltage  AmpGain  Mode
P1     FSC       E03      1.00     Lin
P2     SSC       551      1.00     Lin
P3     FL1       690      1.00     Log

Threshold:
Primary Parameter: FSC
Value: 52

Secondary Parameter: None
  • Raw FCS data files are here


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