Membrane choice & spurious hybridization?
graham at biodec.wustl.edu
Mon Jan 29 17:52:36 EST 1996
Has anyone encountered the suggestion before that "anomalous"
hybridization of defined probes to a variety of targets might be
due to choice of nylon membrane?
I'm probing bacterial genomic cosmid libraries slot blotted onto
MagnaGraph (Micron Seperations) membranes and probed with a
variety of defined DNAs (groEL, sod, etc.) Probes are generated
by either random priming of gel-purified fragments or specifically
priming the ends of gel-purified linearized plasmids.
Why would these specific probes light up much of the cosmid
library at very high stringency conditions? The probes are cloned
sequences Mycobacteria (high GC (60%) organisms). The cosmid
libraries contain Mycobacterial genomic fragments and are grown
in E.coli and prepared for blotting as crude alkaline lysis
minipreps with addition of RNAse but not removal of ribo-oligos.
Please E-mail copies
J. Graham PhD
Washington University of St. Louis
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