Luciferase Assay - Revision history

Davebrid: Changed catalog number

2017-02-27T14:30:39Z

Changed catalog number

← Older revision		Revision as of 14:30, 27 February 2017
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	==Materials==		==Materials==
	*Dual Glo Reporter Assay System (Promega # ~~E1910~~)		*Dual Glo Reporter Assay System (Promega # E2910)
	*To prepare both of these buffers resuspend the lyophylized solution and aliquot in -80		*To prepare both of these buffers resuspend the lyophylized solution and aliquot in -80
	*Dual-Glo Luciferase Buffer		*Dual-Glo Luciferase Buffer

Davebrid at 14:30, 27 February 2017

2017-02-27T14:30:05Z

← Older revision		Revision as of 14:30, 27 February 2017
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	==Materials==		==Materials==
	*Dual ~~Luciferase~~ Reporter Assay System (Promega # E1910)		*Dual Glo 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		*To prepare both of these buffers resuspend the lyophylized solution and aliquot in -80
	*~~Prepare~~ Luciferase ~~Assay Reagent (LARII) by resuspending the lyophilized Luciferase Assay Substrate in Luciferase Assay~~ Buffer ~~II (10ml). Aliquot remaining and store at -70.~~		*Dual-Glo Luciferase Buffer
	*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~~).		*Stop & Glo Buffer
	*~~Plate Reader~~ (~~Book ahead for about 30 min total~~)		*Dual Glo Stop & Glo Substrate (Molecular Biology Stuff at -20)
			*D-PBS
			*Cells transfected with luciferase reporter
			*Tube-based luminometer (GloMax 20/20)


	==Protocol==		==Protocol==
	#Transfect cells with ~~reporter~~ and pRL vector (50:1) and plate in a 96 well ~~white~~ plate		#Transfect cells with 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 plate, but the volumes here are for a 12-well dish
	#Treat cells as required		#Treat cells as required
	#~~Prepare 1X PLB using 5X stock~~ and ~~water~~		#Thaw out enough Dual Glo Buffer and Stop & Glo Buffer for the assay. You will need 100 uL per well of each.
	#Wash wells once with ~~100 ul~~ D-PBS -/-		#Wash wells once with 1 mL D-PBS -/-, aspirate PBS. Can freeze the cells at this point if needed.
	#Add 20 uL ~~PLB~~ to well ~~and incubate~~ on ~~a shaker~~ for ~~15 min~~ at ~~4 degree~~		#Add 100 uL PBS and 100 uL Dual-Glo Buffer to each well
	#~~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~~		#Incubate on rocker for at least 10 minutes
	#Set ~~plate reader~~ to ~~luminesence~~		#Transfer liquid from each well (200 uL) into eppendorf tubes
	#~~Ensure correct measurement head is installed (one light~~ tube~~) and~~ it ~~is set to do a top read~~		#Set luminometer to measure at a 10s integration.
	#~~Set temperature control~~ to ~~off~~		#Measure each tube individually recording the values, or saving it o excel via the GloMax software
	~~#Set plate layout under protocol button for both Luciferase Assay I~~ and ~~Luciferase Assay II~~		#Prepare Stop & Glo reagent by adding the Dual Glo Stop & Glo Substrate to the Stop & Glo Buffer at a 1:100 dilution and mix well
	#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 to each tube and mix
	#~~Add 100 uL of Stop and Glo Reagent and read luminescence again using Luciferase Assay II~~		#Incubate at least 10 minutes
	#Calculate relative luciferase activity by dividing results from Assay ~~I by~~ Assay II		#Measure the Renilla luminesence in the same order
			#Calculate relative luciferase activity by dividing results from the Luciferase Assay over the Renilla Assay

			[[ Category: Luciferase ]]
			[[ Category: Cell Culture ]]
			[[ Category: Tissue Culture ]]
			[[ Category: Promoters ]]
			[[ Category: Transcription ]]

CSherry at 18:35, 25 November 2009

2009-11-25T18:35:03Z

Davebrid at 20:02, 2 June 2009

2009-06-02T20:02:45Z

← Older revision		Revision as of 20:02, 2 June 2009
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	==Protocol==		==Protocol==
	*Transfect cells with reporter and pRL vector (50:1) and plate in a 96 well white plate		#Transfect cells with reporter and pRL vector (50:1) and plate in a 96 well white plate
	*Treat cells as required		#Treat cells as required
	*Prepare 1X PLB using 5X stock and water		#Prepare 1X PLB using 5X stock and water
	*Wash wells once with 100 ul D-PBS -/-		#Wash wells once with 100 ul D-PBS -/-
	*Add 20 uL PLB to well and incubate on a shaker for 15 min at RT		#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		#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		#Set plate reader to luminesence
	*Ensure correct measurement head is installed (one light tube) and it is set to do a top read		#Ensure correct measurement head is installed (one light tube) and it is set to do a top read
	*Set temperature control to off		#Set temperature control to off
	*Set plate layout under protocol button for both Luciferase Assay I and Luciferase Assay II		#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 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		#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		#Calculate relative luciferase activity by dividing results from Assay I by Assay II

Davebrid: initial page

2009-06-02T20:02:23Z

initial page

New page

==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

← Older revision		Revision as of 18:35, 25 November 2009
Line 1:		Line 1:
	==Materials==		==Materials==
	*Dual Luciferase Reporter Assay System (Promega # E1910)		*Dual Luciferase Reporter Assay System (Promega # E1910)
	*Passive Lysis Buffer (PLB at 5X~~; Promega # E1941)~~ at -20		*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
	*Luciferase Assay Reagent (LARII ) at -70~~, use fresh aliquot, thaw at room temp and mix~~		*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		*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)		*Plate Reader (Book ahead for about 30 min total)

Line 12:		Line 12:
	#Prepare 1X PLB using 5X stock and water		#Prepare 1X PLB using 5X stock and water
	#Wash wells once with 100 ul D-PBS -/-		#Wash wells once with 100 ul D-PBS -/-
	#Add 20 uL PLB to well and incubate on a shaker for 15 min at RT		#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 ~~assay~~. Reagent and LARII should be at room temperature		#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		#Set plate reader to luminesence
	#Ensure correct measurement head is installed (one light tube) and it is set to do a top read		#Ensure correct measurement head is installed (one light tube) and it is set to do a top read
	#Set temperature control to off		#Set temperature control to off
	#Set plate layout under protocol button for both Luciferase Assay I and Luciferase Assay II		#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 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		#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		#Calculate relative luciferase activity by dividing results from Assay I by Assay II