genomic DNA contamination using Wizard plasmid kit

Neil Saunders neil.saunders at unsw.edu.au
Tue Jun 5 02:10:11 EST 2001


Dear group,

We are currently using Promega Wizard plasmid miniprep kits to try and
detect plasmids in archaea.  The preps are a bit inconsistent, but we seem
to be seeing a discrete band of around 18-20 kbp.  The problem comes during
restriction digests-under normal conditions we see a smear reminiscent of
chromosomal digest, less enzyme/time seems not to cut at all.

Because of the size and appearance of the band, I wondered if it could be
chromosomal DNA contamination.  Has anyone ever seen this with a Wizard
plasmid prep?  I'd have thought that the precipitation step should remove
all chromosomal, but we are using a lot of cells (low final OD), so I
wondered if overloading could be a problem.  Although the band doesn't look
chromosomal (below the lambda 23 kbp and quite discrete), it doesn't look
quite plasmid either, especially when digested.

thanks for any thoughts,

Neil Saunders

--
School of Microbiology & Immunology,
University of New South Wales,
Sydney 2052,
NSW, Australia

Ph: +61 2 9385 2093
Fx: +61 2 9385 1591

http://www.crosswinds.net/~nfws/index.htm
ArchaeaWeb
http://www.archaea.unsw.edu.au/





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