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G418 selection

01/12/2010
Kristie Nybo

In this Troubleshooting Forum column, a forum user has a problem seeing the difference between untransfected and transfected cells when trying to select stably transfected cells using G418.

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The BioTechniques Molecular Biology Forums brings bench scientists together in an online community to ask for and share advice regarding laboratory problems. This week’s highlighted question comes from the General Methods forum.

Q: I have been transfecting HEK293 cells using lipofectamine2000 from Invitrogen and have had fairly good transient results. My problem is that when I try to select for stably transfected cells using G418 in a range of concentrations from 0.2-2.0mg/ml, I don’t see any difference between untransfected and transfected cells. After a while, I see more survival of cells in the higher G418 concentrations and cells in these cultures begin to float. I haven't seen 100% death of the untransfected cells even in 2 mg/ml G418 for over a week.

Does anyone have any thoughts on why this is happening? Can someone recommend a tried and tested method for selecting for neomycin resistance?

A: Sensitive cells may take 3-4 weeks before being killed. However, 2 mg/ml is high so I'm surprised at your results. Are you using any other antibiotics with your G418? This may interfere. Are you replacing spent media…

A: I just made a stable Hela cell line using a plasmid that encodes neo resistance. After electroporating the cells, I left them in G418-free medium for 24 hours to let the cells recover and then I added fresh medium containing…

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