Changes

*Dual Luciferase Glo Reporter Assay System (Promega # E1910)*Prepare required amount To prepare both of 1X Passive Lysis Buffer (PLB) by adding 1 volume 5X PLB to 4 volumes of distilled water. Mix well these buffers resuspend the lyophylized solution and store at aliquot in -2080*Prepare Dual-Glo 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&*Dual 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 ReagentMolecular Biology Stuff at -20).*Plate Reader D-PBS*Cells transfected with luciferase reporter*Tube-based luminometer (Book ahead for about 30 min totalGloMax 20/20)

#Transfect cells with reporter 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 white plate, but the volumes here are for a 12-well dish

#Prepare 1X PLB using 5X stock Thaw out enough Dual Glo Buffer and waterStop & Glo Buffer for the assay. You will need 100 uL per well of each.#Wash wells once with 100 ul 1 mL D-PBS -/-, aspirate PBS. Can freeze the cells at this point if needed.#Add 20 100 uL PLB PBS and 100 uL Dual-Glo Buffer to each well and incubate #Incubate on a shaker rocker for 15 min at 4 degreeleast 10 minutes#Prepare Stop and Glo Reagent (dilute Transfer liquid from 50X in Stop and Glo Buffer). Need 100 uL per each well in 96-well plate. Reagent and LARII should be at room temperature(200 uL) into eppendorf tubes#Set plate reader luminometer to luminesencemeasure at a 10s integration.#Ensure correct measurement head is installed (one light Measure each tube) and individually recording the values, or saving it is set to do a top reado excel via the GloMax software#Set temperature control Prepare Stop & Glo reagent by adding the Dual Glo Stop & Glo Substrate to off#Set plate layout under protocol button for both Luciferase Assay I the Stop & Glo Buffer at a 1:100 dilution and Luciferase Assay IImix well#Add 100 uL of LARII plate to each tube and then add 20uL of lysates and read luminescence (2s delay, 10s measurement using Luciferase Assay Imix#Add 100 uL of Stop and Glo Reagent and read luminescence again using Luciferase Assay IIIncubate at least 10 minutes#Measure the Renilla luminesence in the same order#Calculate relative luciferase activity by dividing results from the Luciferase Assay I by over the Renilla Assay II [[ Category: Luciferase ]][[ Category: Cell Culture ]][[ Category: Tissue Culture ]][[ Category: Promoters ]][[ Category: Transcription ]]