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transfection efficiency


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#1 Julie123

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Posted 01 May 2010 - 12:45 PM

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

#2 Delta

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Posted 11 May 2010 - 12:33 PM

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-...osts/12250.html

Edited by Delta, 11 May 2010 - 12:33 PM.





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