Technique / Molecular Biology / DNA analysis techniques / DNA southern blot

Plasmid vector unspecific hybridization

Plasmid vector unspecific hybridization

I am screening a plasmid cDNA library of the liver (pSport-vector). After
picking the positives I do minipreps, cut out the inserts from the vector
by RE-digestion and separate by agarose gel electrophoresis. The blotted
gel is hybridized and washed under high stringency conditions (65 C, 0,1x
SSC). On the film I always see hybridization of my probe to the
vector and not as expected only to the positive insert DNA. Sequence
analysis of the vector and the probe show only 30 bp which have 75%
similarity.

How do I get rid of those unspecific hybridizations and what could be the
cause?
Thanks,
Thomas
Original post

Is the hybridisation to your insert stronger than that to the vector? If so you could dilute the digestion products until you only get a signal from the insert.
Original post

Hybridization of my probe to the vector is weaker than to a true positive,
but quite strong, so that I can see clear bands after 2h exposition.
Thanks
Thomas
Original post

the problem is that agarose gels do not do a very good job of separating
the vector from the insert - I had a similar experience - to test things I
digested the plasmid, separated the two fragments (2.3 and 1 kb) on a 1%
gel, cut out each band and purified the DNA by melting the agarose and
phenol extraction - the DNA was then run again on a 1% gel, the bands cut
out and DNA purified and run AGAIN on a 1% gel - this gel was blotted and
hyb'd with the uncut plasmid - both bands showed up in both lanes, and
though the 'wrong' one was quite a bit lighter, it was easily detectable

I have no good explanation for this but have seen it for years

--
David Grant
Original post

Last update 22-Aug-2003, Rating of 0 votes.