G418 selection problems

Have you tried growing your clones at a lower concentration of G418?
When selecting on YE5S/G418 plates we routinely use a concentration of
0.5mg/mL. In liquid culture, however, we drop this 8-fold to an
approximate concentration of 60ug/mL. This, according to a post doc in
our lab (cheers J.P.), is sufficient to kill pombe without the G418
resistance gene (after 2 hours) but high enough to enable selection of
the appropriate clones. I suggest playing around with the
concentration of geneticin using a G418- strain to determine the
optimal amount of G418 required for complete killing, then go back and
test out your G418+ constructs.

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