G418 in plasmid prep

[Martin Kennedy]

In article <CMtBCx.859 at news.cis.umn.edu>, selby at lenti.med.umn.edu (Scott Selby (Med Heme)) writes:
> I have several plasmids with the neo gene in them and would like someone 
> in netland to give me an appropriate concentration of G418 for selection 
> of transformants using these plasmids.  Thanks, in advance.

Depends on the cell line and the batch of G418.  Each batch has a different
proportion of active ingredient.  For mammalian cells, try a range of
100-500ug/ml G418 (final conc in the medium), and use the one that gives
killing of the cells within 3-5 days.  If you're using lymphoid cells, I'd
switch to hygromycin, otherwise you need upwards of 1mg/ml.  Don't forget too
that there are two forms of the neo gene about, one gives better resistance
than the other due to a mutation in the active site (see PNAS 87, p3435;
Yenofsky et al., 1990).

-- 
Cheers,

Martin

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