G418 Stable Transfection - (Sep/06/2005 )

I'm having trouble selecting clones for stable transfection. I have Cos1 cells growing in a 10cm dish and I have been steadily increasing the G418 concentration. It is now up to 2mg and I still have lots of cells. I trypsinized a small area and performed PCR for my protein and it was positive. Is it possible all these cells are positive? Could it be the G418 is not working? I made it fresh with H20 and then filtered. Should I have used PBS?
Thanks

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2 mg/ml G418 is very high! How long have you been selecting?
And by which method did you do your transfection?
Cos-1 cells contain SV40 large T and are very easy to transfect. Don't be surprised if you hit 80% of your cells.

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I've been selecting for 3 weeks now.
I used Lipofectamine/Plus Reagent from Invitrogen.
If its possible all these cells are positive, then I guess I will dilute them very highly and plate and hope to be able to pick up a few single colonies?

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Yes, it is very likely that all your cells are positive. You can simply trypsinise your cells and you have made a polyclonal celline.

To isolate monoclonal cell lines, you will have to repeat the transfection in a less efficient way (e.g calcium phosphate transfection, far less specific DNA, mixed with carrier DNA etc.).

Also include a transfection with a plasmid lacking your selection marker, so that you can follow selection in time.

Good Luck

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Thanks so much for your help!!

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