Luciferase Assay: Difference between revisions

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==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==

#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

#~~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 ]]

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 ==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==
-#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
-#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 ]]