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Team:TUDelft/15th of October protocol
From 2008.igem.org
[http://www.tudelft.nl ]
[http://www.tudelft.nl ]
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.
Measurements
- 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.
