Lidstrom: Tecan Plate Reader

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About the Tecan Infinite f500 plate reader


BCA Assay for Protein concentration

  • Use the 560 nm excitation filter.

Fluorescence-based promoter tests

Basics

  • If you have promoters in front of a fluorescent protein and want to test the promoter strength. The answer you get will depend (nonlinearly!) on:
    • The OD of the sample matters
    • The gain you chose matters
  • Should you subtract out fluorescence of the no-promoter control or divide the test value by the control?
    • Evidence of both can be found in the literature
    • You may have negative values for some samples.
      • Is there a way to blank it??
  • Evidence that the gain and the OD of the sample matter:
    • These data represent the same E. coli line with either an empty vector control or a promoter in front of red fluorescent protein.  The absorbance for each sample has been divided by OD600, then plotted as a function of OD at different gains ranging from 20 to just below saturation for the highest OD samples. The y-scales (y-axes) for each plot are free (different) -JM 8/2013
      These data represent the same E. coli line with either an empty vector control or a promoter in front of red fluorescent protein. The absorbance for each sample has been divided by OD600, then plotted as a function of OD at different gains ranging from 20 to just below saturation for the highest OD samples. The y-scales (y-axes) for each plot are free (different) -JM 8/2013

You should definitely:

  • Compare the test to a control at the same OD.
  • Compare across samples at the same OD
    • If you are comparing different cell types, (e.g. E. coli compared to a small methylotroph) you can't totally compensate for effects of cell size, but using the same OD should help.
  • This will require doing calculations for dilutions. You cannot just assume that 5uL of each type of cells + x uL water/buffer will give you the same OD.
  • Use at least 50 uL (100 is better) so the 96-well plate has enough fluid
  • Use the correct plate. Use one with black-sided wells & plastic that is compatible at your wavelength.

You should probably

  • put cells in a non-fluorescent buffer.
    • LB is known to contain fluorescent molecules.

Sample Protocol

See "Tips for Fluorescence Microplate Assays" in Invitrogen's Fluorescence Microplate Assays, 7th edition

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