Homogeneous plasmid population?

Harry Witchel Harry.Witchel at Bristol.ac.Uk
Tue Nov 11 07:13:43 EST 1997


Hi gang --
   I am transfecting a K+ channel plasmid into mammalian cells.  At
various stages in the plasmid's construction I have sequenced it and
mapped it, and it always appears to be what we expect.  It also makes
nice K+ channel currents, but when we analyse the currents, our
deactivation curve is best fitted by two exponentials. Previous work in
the literature on this channel successfully fitted the data to one
exponential.
   This suggests that we have two channels, not one, in our cells.  Our
cells (CHO) do not normally have K+ channels when not transfected.  We
know that the channels are formed as homotetramers, but heterotetramers
could also form if differing subunits were available..
    One possibility is that my plasmid is really a population of two
plasmids which are similar but not identical.  How do I guarantee that I
have only one plasmid species in my transfections, and how would I
purify out one plasmid if I did indeed have two plasmids in my
population.
    Thanks,
        Harry

--
Harry J. Witchel, Ph.D.
Dept. Physiology
Medical School
Bristol  BS8 1TD
   England

Harry.Witchel at Bristol.ac.uk
http://www.bris.ac.uk/Depts/Physiology/Staff/hw.htm





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