In addition to the ideas here, you might want to try playing with the
optimal FOA concentration. While I have found that 0.2% works most of the
time, sometimes it does result in background growth, depending on what you
have in the strain, and the incubation conditions used.
I've also added 5-FOA powder directly to my autoclaved media, giving the
desired concentration (w/v) once it had cooled to approx 50-55 degrees, and
allowing it to stir in for about 10 minutes. I have found no
contamination problems doing this (I'm quite possibly just extremely lucky),
and is an easy alternative to making two solutions.
"Claudette C." <claudettecoert at yahoo.com> wrote in message
news:b83m6h$avk$1 at mercury.hgmp.mrc.ac.uk...
> Have been trying to use 5FOA to dedelect for URA+ to no avail. I've
> tried adding proline to the media in order to make the cells
> hypersensitive to 5FOA and have also tried replica plating to new 5FOA
> plates after initial growth.
>> The problem is that after each try, instead of only a few cells
> growing, I find a lawn of cells. How many cfu/ml should I be spreading
> in the first place, and also is there anything I might be missing?
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