# Lidstrom: Tecan Plate Reader

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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?? | ||

- | * Check your | + | * Check the linearity of your OD and fluorescence across cell dilutions: |

- | ** Check that OD scales linearly with the amount of cell suspension added | + | ** Check that OD scales linearly with the amount of cell suspension added '''and has intercept = 0''' |

- | ** Check that fluorescence intensity scales linearly with the amount of cell suspension added | + | *** make sure you subtract the OD of the media without cells first. |

- | ** | + | ** Check that fluorescence intensity scales linearly with the amount of cell suspension added '''and has intercept = 0''' |

+ | *** correction via controls may be necessary if the intercept's magnitude isn't negligibly small. | ||

+ | ** If both are linear with intercept = 0, then you know that fluorescence/OD ratio will be a contant-ish number that doesn't depend on the cell concentration. | ||

+ | *** If you divide the line y<sub>1</sub>=slope<sub>1</sub>*x<sub>OD</sub> + b<sub>1</sub> by y<sub>2</sub>=m<sub>2</sub>x<sub>OD</sub>+b<sub>2</sub>, and both b<sub>1</sub> and b<sub>2</sub> are zero, then the x<sub>OD</sub>s cancel out and a constant number is left behind. '''This is the number you want to report.''' | ||

** Then you can calculate fluorescence and summarize your results. | ** Then you can calculate fluorescence and summarize your results. | ||

** Sample data: | ** Sample data: | ||

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## OD absorbance = 540nm (600 is also great) | ## OD absorbance = 540nm (600 is also great) | ||

[[image:2013_12_02 sample fluorescence assay protocol.png|thumb|center|upright=2|2013_12_02 sample fluorescence assay protocol.]] | [[image:2013_12_02 sample fluorescence assay protocol.png|thumb|center|upright=2|2013_12_02 sample fluorescence assay protocol.]] | ||

+ | # I subsequently learned that the z-position can be calculated automatically, meaning it can measure the height of the fluid. |

## Revision as of 22:10, 9 June 2014

Back to Protocols

## Contents |

## About the Tecan Infinite f500 plate reader

- This plate reader can do fluorescence tests.
- Product literature, machine specs.
- The plate reader software writes directly to an excel file you can save. If you want to save the spreadsheet that comes out as a CSV, chose Windows .csv in excel. The default .csv file has ^M as the newline delimeter, but Windows .csv has one line per row in the spreadsheet.

## BCA Assay for Protein concentration

- Use the 560 nm excitation filter.

## Fluorescence-based promoter tests

### Basics

- Use a plate with a flat bottom but black well walls. The black walls makes sure the samples don't interfere with one another. This isn't an issue when excitation isn't used (e.g. absorbance measurements for BCA assay or cell culture density measurement).
- 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??

- Check the linearity of your OD and fluorescence across cell dilutions:
- Check that OD scales linearly with the amount of cell suspension added
**and has intercept = 0**- make sure you subtract the OD of the media without cells first.

- Check that fluorescence intensity scales linearly with the amount of cell suspension added
**and has intercept = 0**- correction via controls may be necessary if the intercept's magnitude isn't negligibly small.

- If both are linear with intercept = 0, then you know that fluorescence/OD ratio will be a contant-ish number that doesn't depend on the cell concentration.
- If you divide the line y
_{1}=slope_{1}*x_{OD}+ b_{1}by y_{2}=m_{2}x_{OD}+b_{2}, and both b_{1}and b_{2}are zero, then the x_{OD}s cancel out and a constant number is left behind.**This is the number you want to report.**

- If you divide the line y
- Then you can calculate fluorescence and summarize your results.
- Sample data:

- Check that OD scales linearly with the amount of cell suspension added

#### 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 Protocols

- See "Tips for Fluorescence Microplate Assays" in Invitrogen's Fluorescence Microplate Assays, 7th edition
- Janet's protocol for RFP: use filters we have, even though they aren't ideal. Wavelengths for fluorescent proteins: here.
- excitation = 535(25) nm. 10 flashes (default setting).
- emission = 612(10) nm. 10 flashes (default setting).
- OD absorbance = 540nm (600 is also great)

- I subsequently learned that the z-position can be calculated automatically, meaning it can measure the height of the fluid.