Difference between revisions of "Luciferase Assay"
From Bridges Lab Protocols
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==Protocol== | ==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 | |
Revision as of 20:02, 2 June 2009
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
