Lidstrom: Tecan Plate Reader

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(New page: Back to Protocols == About the Tecan Infinite f500 plate reader == * This plate reader can do fluorescence tests. * [http://www.tecan.com/platform/apps/product/ind...)
(Fluorescence-based promoter tests)
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*** Is there a way to blank it??  
*** Is there a way to blank it??  
* 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=2.0|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.]]  
+
** [[image:2013_08_fluorescence readings for at different gain values.jpg|thumb|center|upright=3.0|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. -[Users: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.  

Revision as of 14:06, 26 August 2013

Back to Protocols

About the Tecan Infinite f500 plate reader


Fluorescence-based promoter tests

  • 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:
    • JM 8/2013]
      JM 8/2013]
  • You should definitely:
    • 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.
  • Things you should probably do:
    • put cells in a non-fluorescent buffer.
      • LB is known to contain fluorescent molecules.
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