Luciferase Assay: Difference between revisions

Line 8:

==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

@@ Line 8: / Line 8: @@
 ==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