Luciferase Assay: Difference between revisions
Jump to navigation
Jump to search
No edit summary |
Davebridges (talk | contribs) No edit summary |
||
| Line 1: | Line 1: | ||
==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