Luciferase Assay
1. Pick clones and split the same clone into three wells of a six-well
plate. Keep one well for stock cell, another two wells for transfection
2. Transfect two well of cells with pTRE-tight-luc (FuGene Reagent :
DNA =3:2)
3. After 3hr, add Doxycycline ( 1.5ug/ml) to one of the two wells
4. Incubate the transfected cells for 48hr.
5. Remove media, add 1ml 1xPBS, remove PBS
6. Add 1ml 1xPBS , scrape cells (use upside down blue tip) and pipette
off all into 1.5 ml eppendof tube
7. Spin down at 3500 rpm for 5 min at 4°C
8. Keep pellet, frozen if not assaying at same day
9. Lyse cell pellets with 50 ul of luciferase lying buffer, pipette up and
down for 15 times, keep on ice for 15 min
10.Spin down at 11,000 rpm for 10 min
11.Pipette out all supernatant
12.Use 10 ul of supernatant and 100 ul of luciferase assay buffer for
assay
13.Determine the protein concentration for all supernatant ( Don’t add
DTT in lysing buffer for diluting, it will interfere with protein assay )
14.Normalization of luciferase activity with protein concentration