Mo bio question: Selection of bleo resistance

Brett Tyler bmtyler at UCDAVIS.EDU
Sun Feb 19 17:28:49 EST 1995


>I'm hoping some mo bio type in this group can advise me on selection of
>bleomycin resistance in Neurospora. Whenever I transform _N. crassa_ with
>plasmids encoding bleomycin resistance (e.g. pAB520), I get heavy
>background lawns, even though I include the drug in both top and bottom
>agar, can confirm that the selection "works" (untransformed recipient is
>indeed bleo sensitive), have checked the pH of the media, am following the
>suggestions of the original paper on this selection. Anyone have experience
>with this selection working? Thanks in advance. -Peter
>P.S. Contrary to rumor, my brother did _not_ work on the set of The Highlander.

If you send me a detailed write up of your transformation and selection
procedure, I will have a look through it to see if there is anything I can
spot which may improve the selection.

In the meantime, you could simply try increasing the bleomycin
concentration and/or lowering the density of spheroplasts you are plating.
You can actually eliminate the bottom agar altogther if you want to save on
bleomycin.  Are you doing any outgrowth of the transformed sperplasts prior
to plating - this will make them much more resistant.





****************
Brett Tyler
Professor
Department of Plant Pathology
University of California, Davis
bmtyler at ucdavis.edu
****************





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