Luciferase Assay

Materials

Dual Glo Reporter Assay System (Promega # E1910)
To prepare both of these buffers resuspend the lyophylized solution and aliquot in -80
Dual-Glo Luciferase Buffer
Stop & Glo Buffer
Dual Glo Stop & Glo Substrate (Molecular Biology Stuff at -20)
D-PBS
Cells transfected with luciferase reporter
Tube-based luminometer (GloMax 20/20)

Transfect cells with pGL3/4 plasmid and pRL vector (50:1) and plate in a 12 well plate. Can also do these assays in a 48 or 96 well plate, but the volumes here are for a 12-well dish
Treat cells as required
Thaw out enough Dual Glo Buffer and Stop & Glo Buffer for the assay. You will need 100 uL per well of each.
Wash wells once with 1 mL D-PBS -/-, aspirate PBS. Can freeze the cells at this point if needed.
Add 100 uL PBS and 100 uL Dual-Glo Buffer to each well
Incubate on rocker for at least 10 minutes
Transfer liquid from each well (200 uL) into eppendorf tubes
Set luminometer to measure at a 10s integration.
Measure each tube individually recording the values, or saving it o excel via the GloMax software
Prepare Stop & Glo reagent by adding the Dual Glo Stop & Glo Substrate to the Stop & Glo Buffer at a 1:100 dilution and mix well
Add 100 uL to each tube and mix
Incubate at least 10 minutes
Measure the Renilla luminesence in the same order
Calculate relative luciferase activity by dividing results from the Luciferase Assay over the Renilla Assay