Team:Heidelberg/Project/miRNA Kit
From 2010.igem.org
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==Methods== | ==Methods== | ||
- | Dual Luciferase assay | + | === Dual Luciferase assay=== |
We measured the knockdown of firefly luciferase using the Promega Dual Luciferase Reporter Assay. | We measured the knockdown of firefly luciferase using the Promega Dual Luciferase Reporter Assay. | ||
The DLR™ Assay System provides an efficient mean of performing dual-reporter assays, where the activities of firefly (Photinus pyralis) and Renilla (Renilla reniformis) luciferases (RL) are measured sequentially from a single sample. Firefly and Renilla luciferases can be used as a good reporter system, as those two enzymes have dissimilar enzyme structures and substrate requirements. This allows for selective discrimination between their bioluminescent reactions. The firefly luciferase (FL) reporter is measured first by adding Luciferase Assay Reagent II (LAR II) to generate a stabilized luminescent signal. After quantifying the firefly luminescence, this reaction is quenched, and the Renilla luciferase reaction is simultaneously initiated by adding Stop & Glo® Reagent to the same tube. The Stop & Glo® Reagent also produces a stabilized signal from the Renilla luciferase, which decays slowly over the course of the measurement. Here, Renilla luciferase is used for normalization. The measurements were conducted on the Promega GLOMAX 96 Microplate Luminometer using the Promega standard protocol. | The DLR™ Assay System provides an efficient mean of performing dual-reporter assays, where the activities of firefly (Photinus pyralis) and Renilla (Renilla reniformis) luciferases (RL) are measured sequentially from a single sample. Firefly and Renilla luciferases can be used as a good reporter system, as those two enzymes have dissimilar enzyme structures and substrate requirements. This allows for selective discrimination between their bioluminescent reactions. The firefly luciferase (FL) reporter is measured first by adding Luciferase Assay Reagent II (LAR II) to generate a stabilized luminescent signal. After quantifying the firefly luminescence, this reaction is quenched, and the Renilla luciferase reaction is simultaneously initiated by adding Stop & Glo® Reagent to the same tube. The Stop & Glo® Reagent also produces a stabilized signal from the Renilla luciferase, which decays slowly over the course of the measurement. Here, Renilla luciferase is used for normalization. The measurements were conducted on the Promega GLOMAX 96 Microplate Luminometer using the Promega standard protocol. | ||
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- | + | ==== References ==== | |
Bruce A. Sherf, Shauna L. Navarro,Rita R. Hannah and Keith V. Dua l-LuciferaseTM Reporter Assay: An Advanced Co-Reporter Technology Integrating Firefly and Renilla Luciferase Assays. WoodPromega Notes Magazine Number 57, 1996, p.02 | Bruce A. Sherf, Shauna L. Navarro,Rita R. Hannah and Keith V. Dua l-LuciferaseTM Reporter Assay: An Advanced Co-Reporter Technology Integrating Firefly and Renilla Luciferase Assays. WoodPromega Notes Magazine Number 57, 1996, p.02 | ||
- | + | ==== Consumables and Reagents ==== | |
LumaPlate, PerkinElmer, catalogue number 6005630 | LumaPlate, PerkinElmer, catalogue number 6005630 | ||
Promega Dual-Luciferase® Reporter Assay System, catalogue number E1910 | Promega Dual-Luciferase® Reporter Assay System, catalogue number E1910 | ||
- | + | ==== Instruments ==== | |
Promega GLOMAX 96 Microplate Luminometer | Promega GLOMAX 96 Microplate Luminometer | ||
Revision as of 10:43, 24 October 2010
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