Help ! with stable transfection - (Jun/11/2006 )

HI

I am trying a stable transfection in a neuroblastoma cell line. I have cloned my gene into a EGFP-C2 vector and I transfected the cells with lipofectamine for 24 hrs. Then passed the cells into petri dishes with dilutions of 1: 20, 1:40, 1:100 (since my cells grow very fast). After the addition of 1000ug/ml of G418 I don't see any fluorescence ?? I can't identify my positive clones
most of the cells that are glowing are either dead or dying. Should I lower the G418 concentration and try again ?


This is my first try ..I am not sure whats going on ?

Bose.

Let's try to find out what's going on here...

Have you performed a concentration curve to find out which concentration of G418 kills your cells in function of time?
Are you 100% sure your gene of interest is cloned in frame? (or does the C mean you cloned C-terminal of EGFP?)
How long are you selecting with G418? G418 selection takes severall weeks...
Have you performed a positive control transfection (with a GFP reporter construct) to see how efficient your transfection was?
Is your gene of interest potentially toxic to your cells?

Browse through this forum, there's a lot of topics concerning stable transfections where you might find helpfull suggestions.