genomic DNA contamination using Wizard plasmid kit

Sergio sergioal at bbm1.ucm.es
Wed Jun 6 05:28:18 EST 2001



Neil Saunders wrote:

> ""Deanne Bell"" <dbell at qnis.net> wrote in message
> news:200106051527.f55FRQp66026 at ns.qnis.net...
> > I'm not quite clear - are you getting the restriction smear AND the band
> in
> > the same sample or do you get one or the other?
>
> I'm not quite clear either!
>
> When we run the sample without digesting, we get a discrete band at about
> 18-20 kbp.
> If we do a "normal" digest (1 ul enzyme, 1 h), we see a smear reminiscent of
> a chromosomal digest.
> If we use less enzyme or less time, we see no digestion (this last one not
> so relevant, presumably just need to "fine-tune" conditions.)
>
> Basically, the digest makes me think of chromosomal, but the undigested
> sample makes me think of large plasmid, hence our confusion.

I don't know the method you use for youe preps, but i had the same result with a
pseudomonas which actually has no plasmids. Finally, it was ramdonly sheared
chromosomal DNA, which migrates around 21 kb marker in 1% agarose gels. You
could try a PFGE to check the plasmids... as we did.

Sergio





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