Ghost bands on plasmid preps

jw Return.Address at Page.Bottom
Sat Mar 31 19:27:07 EST 2001


This ghost band sounds to me like a small amount of supercoiled DNA
(completely uncut plasmid).  When supercoiled plasmid is run on an agarose
gel, it runs faster than the linearized plasmid.  If this digest was only
done for an hour or so, and with a relatively small amount of enzyme or
large amount of DNA, I would expect to see some uncut plasmid.

One other possibility is that the buffer used for the digest did not have
enough salts for proper BamHI digestion, resulting in a little star
activity, which means the enzyme will also cut at another recognition site
slightly different that the native BamHI recognition site.  BamHI is known
to show this star activity with low ionic conditions, or with high glycerol
concentration greater than 5%.

The quickest way to determine if it is supercoiled (uncut) plasmid is to
just run a small amount of your uncut plasmid in a lane next to the cut
plasmid, and compare the bands.  Chances are, I feel, that this is what you
have observed.

JW
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