LOST PLASMID.....!(can never be found)
udbs061 at bay.cc.kcl.ac.uk
Sun Mar 20 11:14:35 EST 1994
<RROHAN at UMAB.BITNET...> wrote
Dear netters, sorry for any duplication but I believe that my previous posting
was send to the black hole newsgroup.
I have a clone (pGEM7Zf+ vector with an approx 600 bp insert in HB101) that
produces colonies on LB/AMP plates. When we try liquid cultures (LB/AMP) there
I have experienced this on several occassions not only with liquid culture
but also plates.
Ampicillin get degraded very fast during overnight culture at 37 deg c.
If you are not using a fresh frozen stock of amp there wouldn't be any
ampicillin left when added to the media to selecct with. add to that
the old cultures (some clones stay potent for nearly a month and some
lose their viability overnight) from which the media was inoculated
might show a good growth or no growth on overnight cultures. Certainly
there wouldn't be any plasmid of our intersest in the well grown cultures.
This has prompted me to use always
1. fresh stock of ampicillin or the stock kept at -22 c.
2. in case of lost plasmid i would take 1:100 dilution of plasmid DNA
and re tansform and make use of the freshly transformed cultures for
plasmid isolation (mini or maxi prep).
as to the reasons why it happens is a mystery. because some clones behave
properly some don't. I don't think any genaralized reasoning has been
given to this loss of plasmid by any body.
3. As a rule I never prolong my cultures more than 6-8 hours at 37 c
with amp in it.
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