Ghost bands on plasmid preps

R. John Lye rjl6n at virginia.edu
Wed Apr 4 06:53:57 EST 2001


Hi Daniel,

Daniel MacArthur wrote:

> I did run a lane of undigested plasmid along with the various digested
> samples in the original experiment (sorry, I wasn't very clear about this in
> my post). The ghost band is present at essentially identical strength in all
> samples, both digested and undigested.

That sure sounds like the infamous "form 4" DNA.  It is resistant to
most restriction digestion.  Make sure that you're not leaving the
prep in the alkaline lysis step for too long, that's where this band
comes from.  It is irreversibly denatured, and won't cut, but can
actually transform bacteria.  In my experience, keeping the time
from addition of the alkaline SDS to the addition of the neutralising
solution less than five minutes (preferably less than four minutes)
seems to minimize this ghost band.

Good luck,
John




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