Why probe hybridizes to vector?

Marko Rehn rehn at phoenix.oulu.fi
Fri Jul 14 01:45:52 EST 1995

Mic Chaudoir (mic at nwu.edu) wrote:
: In article <biolc4-020795165223 at mac-918.sr2-building.uh.edu>,
: biolc4 at jetson.uh.edu (Wenfu) wrote:

: > When I was using isolated DNA fragment( I am sure no any vector sequence in
: > it)
: > to probe digested plasmids, I found my probe hybridizes to the vector.
: > This kind of thing happen to me several times when I was doing the similar
: > thing. Although it did not matter for my results, I am really curious 
: > about the reasons for.
: > Any idea would be appreciated

: Whenever you gel purify something, a residual amount of other DNA in the
: mixture comes along.  For example, if one cuts out a single band from PKS,
: and gel purifies it, some PKS comes along too.  Usually the amount is so
: small that it makes no difference.  However, the only way to completely
: eliminate it is multiple rounds of gel purification (actually, the
: contamination will still be there, just too small to be detectable by any
: means).  Anyway, practically, I find that this problem is only worth
: mentioning when gel purifying from an overloaded gel.  If you overload the
: gel, you will still get seperation, but a larger amount of the plasmid
: will be carried along.  gel purify smaller amounts at a time, or use
: larger gels, and you should solve the problem.

The other way to avoid vector signal is to make your probe by PCR.

Marko Rehn
Dept. Med. Biochemistry
University of Oulu

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