Unspecific hybridization to plasmid-vector

Thomas Gerloff tgerloff at kpt.unizh.ch
Tue Feb 27 09:58:04 EST 1996


Hello netters,

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
  
  



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