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.
• The plate reader software writes directly to an excel file you can save. If you want to save the spreadsheet that comes out as a CSV, chose Windows .csv in excel. The default .csv file has ^M as the newline delimeter, but Windows .csv has one line per row in the spreadsheet.

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??
• Check your data:
  • Check that OD scales linearly with the amount of cell suspension added
  • Check that fluorescence intensity scales linearly with the amount of cell suspension added
  • Then you know that fluorescence/OD should scale linearly with uL of cells
  • Then you can calculate fluorescence and summarize your results.
  • Sample data:

    • 

      Quality check data.

    • 

      Results.

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 Protocols

1. See "Tips for Fluorescence Microplate Assays" in Invitrogen's Fluorescence Microplate Assays, 7th edition
2. Jane'ts protocol for RFP: use filters we have, even though they aren't ideal.
   1. excitation = 535(25) nm. 10 flashes (default setting).
   2. emission = 612(10) nm. 10 flashes (default setting).
   3. OD absorbance = 540nm (600 is also great)

2013_12_02 sample fluorescence assay protocol.