Promega Dual-Luciferase Assay PLB Method
Method-Remove the media from each well using a 1ml pipette to ensure complete removal.
-Add (X)ul of 1x PLB to each well and leave for 3 minutes. (1X Promega passive lysis buffer
(PLB), made by mixing for example 1ml of 5X PLB with 4ml of sterile deionised H2O.)
(X) – 6 well plate- add 200ul to each well, 24 well plate- add 100ul to each well.
-Using a cell scraper, scrape the bottom of the well and then push all the cell debris down to
one corner of the well. Leave the plate sat at an angle to allow all the PLB to flow into the
corner of the well.
-Transfer all the lysate to an Eppendorf tube. At this point the protocol can be stopped and
the lysate stored at -20oC.
-Vortex each lysate sample at max speed for 15 seconds and then centrifuge the samples at
12,000 RPM for 15 seconds. A pellet should form at the bottom. At this point you can
transfer the lysate to a different Eppendorf or avoid taking up the pellet when adding the
sample to the luminometer.
Obtaining ResultsWhen ready to assess the RLU of the sample, bring all reagents to room temperature for at
least 1 hour before starting.
First start by testing the RLU of the empty tube before adding 20ul of sample and then add
100ul of LAR luciferase reagent (Do not wear gloves). Flick the tube 7 times and add it to the
luminometer. If the luminescence of the sample is weak, this value may be deducted to get a
more representative RLU value.
If recording S+G values, take the tube out of the luminometer and add 100ul of Stop and Glo
to the tube. Vortex the sample for 7 seconds and then add the tube to the luminometer.
Empty S+G has a base value of 100-150 RLU.
Return unused samples to -20oC for possible repeat assays.