szat at ERE.UMontreal.CA
Mon Jan 22 16:39:04 EST 1996
In article <1996Jan18.062544.13461 at alw.nih.gov> bernard at elsie.nci.nih.gov (Bernard Murray) writes:
>In article <4dirb7$i4j at scotsman.ed.ac.uk>, chrisb at hgu.mrc.ac.uk says...
>[snip: (dam-, dcm- GM2163 doesn't appear to work)]
>>Someone else mentioned that you need to propagate GM2163 on
>>chloramphenicol to maintain the dam mutation (which is caused by a Tn9
>>insertion). I disagree. The frequency of spontaneous perfect excision
>>of Tn9 is negligibly low.
>Yep, that was me but I was only following NEB's advice. I'm
>glad to hear that you don't have to be as careful.
> Can you (or anyone else who's familiar with transposons)
>comment on the stability of the others used in host strains?
>(eg. the Tn10 tetR in many F' factors and the Tn5 kanR used to
>select for other genotypes). I realise that growing the bugs in
>the presence of tet is necessary/advisable where Tn10/F' is
>required but is it necessary to keep up kan selection when the
>host has a Tn5 genomic marker? At the moment I select my competent
>cells but don't always include kan in the maxiprep cultures (and
>can't say that I've seen anything bad happening).
> Also, does anyone select for the gyrA96 property - and how?
> Just wondering....
>Bernard Murray, Ph.D.
>bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)
Most, if not all transposon insertions in the chromosome are pretty stable,
and selection is certainly not required, though it is handy to use when
you want to make sure that your strain still has the required mutation.
I believe the gyrA96 marker encodes resistance to naladixic acid, though
I may be wrong about this.
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