Difference between revisions of "Luciferase Assay"

Revision as of 20:02, 2 June 2009

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

• 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 RT
• 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
• 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 to 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