IGEM:Harvard/2007/Laboratory Notebooks/Quorum Sensing/Plate Reader Protocol

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Written by Stephanie Lo, July 2007

  1. Turn on the Plate Reader (near the cell culture room) and ensure that the temperature is set at 37C. SoftMax is the program on the nearby PC that controls these settings, though many can be adjusted on the machine itself.
  2. Use a 96-well plate (blk/clr bottom for fluorescence and OD; if OD only, clr bottom is fine).
  3. Add 200uL of sample. Usually, these are straight cells (no dilution). Make sure you pipet up and down the liquid culture before adding anything, so that an accurate and well-mixed sample is added.
    1. Try to put the cells in adjacent wells, since the plate reader can only read rectangular areas.
  4. Insert plate into plate reader, with A1 in the top left corner, without the lid.
  5. Turn on SoftMax.
    1. Check temperature is still at 37C.
    2. Press "Setup", which should be in the default window.
    3. Choose Kinetic and OD.
    4. Wavelength = 600
    5. Choose a time duration. The reader uses the format 00:00:00, where numbers correlate to "hours:min:sec". For OD during growth, before induction, I suggest setting this to 03:00:00 (3 hours), since the program can be stopped early if necessary.
    6. Choose a time interval. I suggest 10 or 15 minutes (00:10:00 or 00:15:00).
    7. Choose premix plate (3 or 5 seconds) and "mix between reads" for 300-500 seconds.
    8. Choose cuvette reference and make sure an LB cuvette is in the cuvette slot in the plate reader.
    9. Turn Pathcheck on
    10. Choose which wells to read
  6. Save preferences, then start the program by clicking "run" (near the top of the window). The machine should start making buzzing noises and the timer near the top of the page will start running.
  7. Check on the OD every once in a while by double-clicking any of the windows. A graph will appear. You can click "auto-scale" to get a better idea of what your OD is. For induction, you may want to wait until OD reaches about 0.3 (should start at about 0.15).
  8. When the OD reaches the desired number, stop the program by clicking the "stop" button (where "run" used to be). Retrieve the plate by pressing the "drawer" button on the machine.
  9. Induce desired samples by adding OHHL.
  10. On plate reader "setup", change to "fluorescence" reading. For GFP/YFP samples, I typically use excitation wavelength: 485 and emission: 525.
  11. For our protocol, I'd leave the fluorescence running for 5 hours minimum. Maybe, if we leave it overnight, we should just leave it running throughout the night (15 hours?) so that the computer doesn't restart itself, etc.
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