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.

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.