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

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 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] BBF Standards: Promoter Measurement Test
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2008-04-02
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
 * 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):

Threshold: Primary Parameter: FSC Value: 52

Secondary Parameter: None
 * Raw FCS data files are here


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