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
- 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
