Team:Tuebingen/Notebook/Protocols/tecanreader
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Using the Tecan Reader
The Tecan reader is used for flourescence spectroscopy. Thus, tested cells should express some sort of flourescent protein. We mainly tested yeast cells that express mOrange (BBa_E2050).
Procedure
- Inoculate 5 mL liquid YPD medium with yeast cells. Create 1:10 and 1:100 dilutions of this liquid culture. Incubate all cultures at 30°C over night.
- On the next day, check OD600 (culture density). Ideally, OD600 is between 1 - 1.5 (maximum: 2.0).
- Transfer cells to Eppendorf tubes and centrifuge at 13 000 rpm for 30 sec.
- Discard supernatant and add 1 mL Aqua dest. Resuspend cells.
- Inoculate 1/3 of initial culture volume (minimum: 600 µL) liquid YPD (OD600 should be between 3 - 5) with cells from previous step.
- Transfer cells to a cuvette, dilute 1:10 with Aqua dest., and measure exact OD600 (necessary for standardization of measured data).
- Transfer 150 µL cell suspension in a black 96-well-plate. Create a 150 µL water blank.
- Measure cells in Tecan-Reader. Adjust settings according to your requirements.
Our settings:- Plate: Greiner 96 Flat Bottom Black [half area]
- Mode: Flourescence intensity
- Excitation: 548 nm
- Emission: 581 nm
- Gain: 150
- Number of flashes: 25
- Time of integration: 25 µs
- If required record excitation spectrum.
Our settings:- Plate: Greiner 96 Flat Bottom Black [half area]
- Mode: Flourescence Scan
- Excitation: 300 - 550 nm
- Step size: 5 nm
- Emission: 581 nm
- Gain: 150
- Number of flashes: 25
- Time of integration: 20 µs
- Standardize measured data to OD600 = 1.0.
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