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
Finland



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