Lidstrom: Tecan Plate Reader

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(Fluorescence-based promoter tests)
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* Evidence that the gain and the OD of the sample matter:   
* Evidence that the gain and the OD of the sample matter:   
** [[image:2013_08_fluorescence readings for at different gain values.jpg|thumb|center|upright=4|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) -[[User:Janet B. Matsen|JM 8/2013]]  ]]  
** [[image:2013_08_fluorescence readings for at different gain values.jpg|thumb|center|upright=4|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) -[[User:Janet B. Matsen|JM 8/2013]]  ]]  
-
* You should definitely:
+
==== You should definitely: ====
-
** Compare the test to a control at the same OD.  
+
* Compare the test to a control at the same OD.  
-
** 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.
+
* Compare across samples at the same OD
-
* Things you should probably do:
+
** 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.
-
** put cells in a non-fluorescent buffer.
+
* 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.
-
*** LB is known to contain fluorescent molecules.
+
* 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 ===
=== Sample Protocol ===
See "Tips for Fluorescence Microplate Assays" in Invitrogen's [http://probes.invitrogen.com/media/publications/108.pdf Fluorescence Microplate Assays, 7th edition]
See "Tips for Fluorescence Microplate Assays" in Invitrogen's [http://probes.invitrogen.com/media/publications/108.pdf Fluorescence Microplate Assays, 7th edition]

Revision as of 14:18, 26 August 2013

Back to Protocols

Contents

About the Tecan Infinite f500 plate reader


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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