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25th of September protocol
From 2008.igem.org
[http://www.tudelft.nl ]
[http://www.tudelft.nl ]
25th of September Luciferase assay protocol
This protocol has been discarded due to interfering of Promega lysis buffer with the protein measurements.
Cell preparation
- Pellet 1 ml of grown culture when the culture is around 0.6 by centrifuging 5 min at 10000 rpm in a tabletop centrifuge.
- Lyse the cells by adding 1x lysis buffer from the Renilla Luciferase Assay kit.
- 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.
