Transient transfection of linearized plasmids - (Jul/02/2013 )
I'm trying to transiently transfect HeLa with linear plasmids (pgl3 backbone, cut at MCS before the enhancer) for a luciferase assay.
I did one round of transfection with these plasmids (both circular and linearized) with extreme gene HP reagent in a 96 well; transfection efficiency was good for circular plasmids, and almost zilch for the linearized plasmids. I used 200ng of the test firefly plasmids, and 5ng of renilla in a co-transfection. I used a dual luciferase kit for assaying the luminescence, following the manufacturer's protocol. As I said, I got a good set of readings for both firefly and renilla (between 100,000- 10,00,000 RLU) in the cells transfected with circular plasmids, and extremely low (less than 2000 RLU) for the linearized plasmids.
Could you please help troubleshoot the problem?
Try linearizing in some area of the plasmid that doesn't include important bits...
Actually I linearized them before the enhancer at the MCS which shouldn't affect the expression.
If you cut at the MCS of pGL3, then you cut in a region that probably includes the binding site for transcriptase and potentially you may have cut the background signal reduction site.
Try cutting somewhere in the Ori, and see what happens.