Why probe hybridizes to vector?

Peter Kuhnert kuhnert at vbi.unibe.ch
Thu Jul 13 06:13:39 EST 1995


amarion at moose.uvm.edu (Amy Marion) wrote:
>
>I routinely produce Southern probes by PCR using "insert-primers" to eliminate
>the presence of vector sequences.  However, I STILL see strong hybridization
>of this probe to vector (specifically Bluescript) sequence.  I believe this is
>causing serious problems since I am trying to use this probe to screen a cDNA
>library constructed in Lambda Zap II (ie., Bluescript).
>
>Any suggestions?
>
>Amy Marion
>marion at smtplink.ipfw.indiana.edu


We fight with the same problem: trying to detect genes for bacterial virulence factors in a wide range of bacterial genera, we label cloned virulence genes. In many cases the negative control strain E.coli K12 becomes a very "virulent" strain due to crosshybridization of plasmid sequences that contaminated the labeling. We tried several approaches. The sequential purification of the template over gels, followed by RP or PCR labelling works in some cases. I am wondering if purification e.g. by HPLC would result in cleaner templates. Did anybody try it?

Peter




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