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.

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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

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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

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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.

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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!

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I'd like to know, whats the cell concetration i must seed on a 6 well plate to get the G418 inhibitory concentration.

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hi
that depends of your cell type. Hek cells can be very diluted but somes cannot support too many dilution.
20% confluency is a good compromise.

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We use 2 mg/ml for the initial selection pressure on HEK293 cells. This is close to the top end of the kill curve. Normally we use 100-400 ug/mL to maintain the selection pressure although at 100 the cells will sometimes lose expression. I would suggest 200-400 ug/mL to maintain the selection.

Good Luck

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