DNA probes for use with prokaryote DNA in Southerns

Douglas Easton dpeaston at wzrd.com
Thu Dec 19 20:14:12 EST 1996


Douglas Easton wrote:
> 
> My usual procedure for making probes from plasmid clones is to cut the
> insert from the vector and gel purify the insert for labeling and use as
> a probe for Northerns or Southerns. Recently I have atttempted to
> identify prokaryotic homologues to eukaryotic sequences. I find that
> even after 2 cycles of gel purification, the vector sequences seem to
> hybridize to the 1 KB ladder that I use in my gels. This ladder is a
> concatemer of a PBR/PUC derived plasmid. The vector I am using is
> XL-Blue.
> 
> Any suggestions for avoiding this problem?
> 
> -Doug Easton
I need to correct myself. The hybridization to the ladder
suggests to me that PBR-like sequences in the procaryotic
DNA, that is being probed for a eukaryotic sequence, may account
for the bands I see in the southern. This would be a serious artefact (a
false positive).

-doug



More information about the Methods mailing list