Team:Tuebingen/Notebook/Protocols/tecanreader

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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

  1. 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.
  2. On the next day, check OD600 (culture density). Ideally, OD600 is between 1 - 1.5 (maximum: 2.0).
  3. Transfer cells to Eppendorf tubes and centrifuge at 13 000 rpm for 30 sec.
  4. Discard supernatant and add 1 mL Aqua dest. Resuspend cells.
  5. Inoculate 1/3 of initial culture volume (minimum: 600 µL) liquid YPD (OD600 should be between 3 - 5) with cells from previous step.
  6. Transfer cells to a cuvette, dilute 1:10 with Aqua dest., and measure exact OD600 (necessary for standardization of measured data).
  7. Transfer 150 µL cell suspension in a black 96-well-plate. Create a 150 µL water blank.
  8. 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
  9. 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
  10. Standardize measured data to OD600 = 1.

 

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