Difference between revisions of "Luciferase Assay"
From Bridges Lab Protocols
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==Materials== | ==Materials== | ||
| − | *Dual | + | *Dual Glo Reporter Assay System (Promega # E1910) |
| − | * | + | *To prepare both of these buffers resuspend the lyophylized solution and aliquot in -80 |
| − | * | + | *Dual-Glo Luciferase Buffer |
| − | *Stop | + | *Stop & Glo Buffer |
| − | * | + | *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 | + | #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 | ||
| − | # | + | #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 | + | #Wash wells once with 1 mL D-PBS -/-, aspirate PBS. Can freeze the cells at this point if needed. |
| − | #Add | + | #Add 100 uL PBS and 100 uL Dual-Glo Buffer to each well |
| − | # | + | #Incubate on rocker for at least 10 minutes |
| − | #Set | + | #Transfer liquid from each well (200 uL) into eppendorf tubes |
| − | # | + | #Set luminometer to measure at a 10s integration. |
| − | # | + | #Measure each tube individually recording the values, or saving it o excel via the GloMax software |
| − | + | #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 | + | #Add 100 uL to each tube and mix |
| − | # | + | #Incubate at least 10 minutes |
| − | #Calculate relative luciferase activity by dividing results from Assay | + | #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 ]] | ||
Revision as of 14:30, 27 February 2017
Materials
- Dual Glo Reporter Assay System (Promega # E1910)
- To prepare both of these buffers resuspend the lyophylized solution and aliquot in -80
- Dual-Glo Luciferase Buffer
- Stop & Glo Buffer
- 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 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
- 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 1 mL D-PBS -/-, aspirate PBS. Can freeze the cells at this point if needed.
- Add 100 uL PBS and 100 uL Dual-Glo Buffer to each well
- Incubate on rocker for at least 10 minutes
- Transfer liquid from each well (200 uL) into eppendorf tubes
- Set luminometer to measure at a 10s integration.
- Measure each tube individually recording the values, or saving it o excel via the GloMax software
- 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 to each tube and mix
- Incubate at least 10 minutes
- Measure the Renilla luminesence in the same order
- Calculate relative luciferase activity by dividing results from the Luciferase Assay over the Renilla Assay
