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