Contaminating plasmid

R. A. KELLN KELLN at Meena.CC.URegina.CA
Thu Jan 13 12:39:13 EST 1994


In <2h165l$29g at mserv1.dl.ac.uk> ray at leicester.ac.uk writes:

> A number of us have had problems with a plasmid which seems to
> contaminate our plasmid stocks. It's ampicillin resistant and
> has a linear size of around 2.2 kb. The strangest aspect is the
> lack of sites for common restriction enzymes. It has an SstI (SacI)
> site but lacks sites for BamHI, HindIII, KpnI, PstI, EcoRI, SalI
> and EcoRV.
> 
> It often appears as a faint band in preps of plasmid DNA from
> constructs in pUC and Bluescript type vectors.
> 
> Can anybody suggest what it might be? I'd be very grateful for
> suggestions.
> 
>   ----------------------------------------------------------------------------
>   Dr Raymond Dalgleish,          JANET: ray at uk.ac.le
>   Department of Genetics,     INTERNET: ray at le.ac.uk
>   University of Leicester,    ICBM NET: 52 37' 23" N, 01 07' 24" W
>   University Road,                 Tel: (+44) 0533 523425
>   Leicester LE1 7RH,               Fax: (+44) 0533 523378
>   United Kingdom.                  
>   ----------------------------------------------------------------------------
>   #include <std_disclaimer.h>
>   ----------------------------------------------------------------------------
>  
>How sure are you that it is a contaminating plasmid per se?  Given the
frequency of this happening to others who are using pUC, I can't help but
wonder if the commonality here is that you are all using an alkaline rapid
lysis procedure and what you are seeing is, in fact, denatured supercoiled DNA
which is refractory to digestion by most restriction enzymes.  If you were to
cut this band from the gel and transform an E.coli and examine the
transformants you would of course find pUC if my suggestion has any merit.  We
ran into these denatured supercoils several years back when doing subcloning
by purifying DNA fragments cloned in pUC's on gels and then trying to clone to
another vector.  To our surprise, we had numerous transformants harboring the
original pUC plasmid.  If you are not using an alkaline lysis procedure, then I
have no idea why you are getting this alien plasmid.  Comments anyone? 



More information about the Methods mailing list