Selection of clones during transfection - Concentration of G418 to be added to the cell. (Aug/20/2004 )

Hi ,

How should one can decide the optimal concentration of geneticin to select a clone during stable transfection if it's not known at what concn. cells will die.

In our lab, we use 0.8mg of G418 (geneticin) per ml of medium for selection, and we lower that to 0.4mg/ml to maintain the selective pressure after that. We work aon the HEK model. I don't know though if the same concentrations apply to other types of cell.

Hope I could help!

Simon

Hi,
can you find out at what concentration cells will die? Construct a kill curve? It takes about 2weeks.
Otherwise 0.4-0.8mg/ml seems to be the range for most cell lines I use

Thanx for the response U have given to me.

But, these cells I'm working with are very sensitive, so can I start with 50ug/ml concn.But then my worry is Cells may loose the transfected DNA if selection pressure is too low.

Hello again,
if you say your cells are sensitive then you can start with a low concentration of antibiotic. Theoretically for a sensitive cell line a small amount of it should still be enough to maintain a selective pressure. You can always run a control non-transfected well, to make sure you are not getting random mutants. If you have the option I'd still try a kill curve, it might actually save you time and cells in the long run.
Good luck!