transfection efficiency - (May/01/2010 )

Hello,

Does anyone have a detailed protocol for monitoring transfection efficiceny using GFP and/or luciferase?I am transfecting cells with siRNA but i dont see good knockdown.Has anyone used lipofectamine2000 with HeLa to cotransfect siRNA and plasmid?what concentrations worked?thx

I'm not sure what you mean by a detailed protocol, but I've had good luck using the reporter assay kits from Promega to detect luciferase/Bgal expression.

For GFP I've typically used florescence microscopy in comparison with a nuclear marker (DAPI, etc) to visualize expression and do cell counts from multiple random fields (# of cells expressing GFP / total # of viable cells = transfection efficiency) . Flow cytomtery is probably the best way to do it if you can.

Here is a similar post from a while back:
http://www.protocol-online.org/biology-for...osts/12250.html