Plasmid DNA Purification

James F Rice jrice at utkvx.utk.edu
Tue Jan 18 11:22:00 EST 1994


	Recently I have been having problems isolating and purifing a 20Kb
plasmid.  I have successfully used standard mini-prep protocols to verify that
a cloning experiment was successfull, and I had no problems isolating the
plasmid DNA using Promega's mini-prep protocol.  I used restriction digest to
verify that the plasmid was the correct one (BamH1, Spe I, and Pst I). 
However, When I attempted to perform a large scale Plasmid Prep. I found that
the DNA I obtained would not cut, using Bam H1, EcoR1, Hind III, Pst I, Spe I,
or Xba I. The Restriction map specifies that all of these enzymes should be
able to cut. The plasmid has been purified through a CsCl/EtBr gradient.  My
first response to this problem was to reprecipitate the prep, followed by
thorough dialysis, in order to remove any contaminants. This did not help
however. I have done many plasmid preps and never encountered this problem. I
then began a repeat of the plamid extraction, but it appears that I may
encounter the same problem.  The plasmid was cloned into E.coli DH5 alpha
cells, and the selection was for tet resistance at 28 ug/ml. I have tried
adding DNA from another P. prep that I have done to see if there is some
contaminant being carried along, but the added DNA cut fine.  The plasmid I am
Having problems with is running at 20Kb on my agarose gels; in a discreet band.  Ha
anyone had a similar problem?  I would welcome any help.  

Thanks.  

---------------------------------------------------------------------------------
	EMail:  JRICE at UTKVX.UTK.EDU
	James Franklin Rice
	Center For Environmental Biotechnology
	10515 Research Drive
	Knoxville, TN 37932-2572
	(615)974-8073












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