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
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transfection efficiency
Started by Julie123, May 01 2010 12:45 PM
1 reply to this topic
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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
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.
