Lidstrom: Tecan Plate Reader: Difference between revisions
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== Fluorescence-based promoter tests == | == 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: | * 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 OD of the sample matters | ||
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*** LB is known to contain fluorescent molecules. | *** 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 11:11, 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
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:
- 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.
Sample Protocol
See "Tips for Fluorescence Microplate Assays" in Invitrogen's Fluorescence Microplate Assays, 7th edition