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檢測熒光素酶
Introduction
Luciferase can be used as a reporter gene to measure the activity of promoters, and/or the transfection efficiency.
Aims
You will be provided with the HEK293/COS cells that you transfected on Monday. Working in groups of two, take the two samples transfected by your group and assay for luciferase activity. Note, in all cases, the total amount of DNA used for the transfection is 2.0µg.
Luciferase assay
Materials required
1 x CCLR （cell lysis reagent） Luciferase Assay Reagent
1 x CCLR （cell lysis reagent） Luciferase Assay Reagent
Experimental procedure
1. Remove the growth medium from the cells to be assayed. Rinse the cells twice in PBS buffer, being careful not to dislodge any of the cells.
2. Add a minimal volume of 1 x CCLR to cover the cells. （250 µl for a 60 mm dish） and incubate for a short period at room temperature.
3. Scrape the attached cells free from the culture dish and transfer the cells and solution to a microcentrifuge tube. Centrifuge for 5 sec at 12,000 rpm to pellet the cell debris.Transfer the supernatant （cell extract） to a new tube and discard the pelleted cell debris
4. Mix 20 µl of room temperature cell extract with 100 µl room temperature Luciferase Assay Reagent. Place the reaction in a luminometer.
5. Measure the light produced for 10 seconds.
6. Record data
Results Ð transfected cells
Record the activity of your groups's transfection in light units （LU）. The results of all the transfections will be discussed when all the data has been compiled.
Discussion
Althougth data interpretation is somewhat subjective, this practial should demonstrate the relative efficiency of the three reagents.
Luciferase Ð Appendix
Controls
To be useful for quantification of gene activity, a number of controls are necessary. The LU values must be correct for cell number and/or total amount of protein. A second luciferase gene can be used as an internal control. Full details can be found on Promega's web site.
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