Team:TUDelft/15th of October protocol


15th of October luciferase assay protocol

Cell preparation

  • Pellet 750 ul of grown culture when the culture is around OD=0.9 by centrifuging 5 min at 10000 rpm in a tabletop centrifuge.
  • Resuspend the cells in 1x PBS.
  • Lyse the cells by 2 times 15 second sonication at max amplitude, with a 15 second interval.
  • Freeze the cells at -20 until measurements will be done.


  • Mix 1 ul of 100X luciferase substrate in 100 ul of assay buffer per sample in the luminometer's reagent tube no. 1
  • Put 20 ul of lysed cells in a white 96 wells plate.
  • Put the prime plate in the luminometer (usually on top)
  • Put the tube with assay buffer under reagent needle no. 1, make sure the tip of the needle is in a position to reach all the assay buffer.
  • Prime the luminometer with 1000 ul assay buffer in the priming plate(make sure the tubing is rinsed before)
  • Measure luciferase activity by:
    • Adding 100 ul of assay buffer to a well
    • Wait 2 seconds
    • Integrate luminescence for 10 seconds.
    • Repeat for every well
    • There is a standard protocol on the computer in which you only have to indicate the wells to be assayed.
  • Purge the tubing (Assay buffer can be stored and frozen for short periods (1 week at most) according to the technical manual)
  • Rinse the tubing with 5000 ul of ethanol, and purge it.
  • Rinse the tubing with 5000 ul of H2O, and purge it.
  • The tubing and injector should be clean and empty now.