Membrane choice & spurious hybridization?

James Graham graham at biodec.wustl.edu
Mon Jan 29 17:52:36 EST 1996


Hello Bionetters,

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

Jim
J. Graham PhD 
Biology Department 
Washington University of St. Louis 



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