Lidstrom: Tecan Plate Reader: Difference between revisions
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** You may have negative values for some samples. | ** You may have negative values for some samples. | ||
*** 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: -[Users:Janet B. Matsen|JM 8/2013] | ||
** [[image:2013_08_fluorescence readings for at different gain values.jpg|thumb|center|upright= | ** [[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)]] | ||
* 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 11:07, 26 August 2013
Back to Protocols
About the Tecan Infinite f500 plate reader
- This plate reader can do fluorescence tests.
- Product literature, machine specs.
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: -[Users:Janet B. Matsen|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.
- put cells in a non-fluorescent buffer.