Luciferase Assay

Materials

• Dual Luciferase Reporter Assay System (Promega # E1910)
• Passive Lysis Buffer (PLB at 5X; Promega # E1941) at -20
• Luciferase Assay Reagent (LARII ) at -70, use fresh aliquot, thaw at room temp and mix
• Stop and Glo Reagent and Buffer at -20
• Plate Reader (Book ahead for about 30 min total)

Protocol

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