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==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
*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