Dual Luciferase Reporter Protocol

We now are using the Dual-Glo system, but for reference this was the protocol using the previous protocol with the plate reader.

Materials

Dual Luciferase Reporter Assay System (Promega # E1910)
Prepare required amount of 1X Passive Lysis Buffer (PLB) by adding 1 volume 5X PLB to 4 volumes of distilled water. Mix well and store at -20
Prepare Luciferase Assay Reagent (LARII) by resuspending the lyophilized Luciferase Assay Substrate in Luciferase Assay Buffer II (10ml). Aliquot remaining and store at -70.
Stop and Glo Reagent and Buffer at -20. Prepare required amount of Stop&Glo Reagent from 50X Stop&Glo Substrate. Add 50X Stop&Glo Substrate to final 1X concentration (i.e. 0.2ml of 50X Stop&Glo Substrate to 10ml of Stop&Glo Buffer to make a 1X solution of Stop&Glo Reagent).
Plate Reader (Book ahead for about 30 min total)

Transfect cells with reporter and pRL vector (50:1) and plate in a 96 well white plate
Treat cells as required
Prepare 1X PLB using 5X stock and water
Wash wells once with 100 ul D-PBS -/-
Add 20 uL PLB to well and incubate on a shaker for 15 min at 4 degree
Prepare Stop and Glo Reagent (dilute from 50X in Stop and Glo Buffer). Need 100 uL per well in 96-well plate. Reagent and LARII should be at room temperature
Set plate reader to luminesence
Ensure correct measurement head is installed (one light tube) and it is set to do a top read
Set temperature control to off
Set plate layout under protocol button for both Luciferase Assay I and Luciferase Assay II
Add 100 uL of LARII plate and then add 20uL of lysates and read luminescence (2s delay, 10s measurement using Luciferase Assay I
Add 100 uL of Stop and Glo Reagent and read luminescence again using Luciferase Assay II
Calculate relative luciferase activity by dividing results from Assay I by Assay II