Lidstrom: Tecan Plate Reader

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(Fluorescence-based promoter tests)
(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=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]]]  
+
** [[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.]]  
* 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 14:07, 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:
    • 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.
      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.
  • 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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