Lidstrom: Tecan Plate Reader

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About the Tecan Infinite f500 plate reader

• This plate reader can do fluorescence tests.
• Product literature, machine specs.

BCA Assay for Protein concentration

• Use the 560 nm excitation filter.

Fluorescence-based promoter tests

Basics

• Use a plate with a flat bottom but black well walls. The black walls makes sure the samples don't interfere with one another. This isn't an issue when excitation isn't used (e.g. absorbance measurements for BCA assay or cell culture density measurement).
• 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

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