LOST PLASMID.....!(can never be found)

Martin Kennedy mkennedy at chmeds.ac.nz
Wed Mar 30 19:54:09 EST 1994


In article <1994Mar20.161435.2507 at bay.cc.kcl.ac.uk>, udbs061 at bay.cc.kcl.ac.uk (MAHARAJAH) writes:
> <RROHAN at UMAB.BITNET...> wrote

lots of stuff deleted.......

> 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

This might be quick, but it is where a lot of these problems come from!!!!  Any
transformation mix selected with ampicillin contains oodles of Amp sensitive
cells, ready to leap into log phase growth the moment all the amp disappears
from a culture.  They aren't carrying the metabolic load of a multicopy
plasmid, so they grow a lot faster and take over your culture.  If you plan to
make a maxiprep, or you have this kind of problem, streak the resistant
colonies out onto another amp plate, so that you get single pure colonies. 
This is a procedure known to all ancient microbiolgists, as it leads to a
****PURE**** colony of cells.  This is something we often overlook, often get
away without doing (because it saves a whole day) but if you aren't aware of
the problem it often causes grief.   Just remember, when you plate a 
transformation mix, for every competent cell that takes up the plasmid there 
are many thousands more that don't.  THESE DON'T DIE, THEY JUST HANG AROUND, 
WAITING FOR THE OPPORTUNITY TO SCREW UP YOUR EXPERIMENTS!!!!!!!!!!!!!!!!!

Cheers,

Martin

NNNN   NN  Martin A Kennedy (E-mail = mkennedy at chmeds.ac.nz)  ZZZZZZZ  
NN NN  NN       Cytogenetic and Molecular Oncology Unit          ZZZ
NN  NN NN           Christchurch School of Medicine            ZZZ
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