荧光素酶检测

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

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