Library Screening Weirdness

Joseph C. Bagshaw jbagshaw at wpi.edu
Thu Aug 24 07:04:23 EST 1995


Robert,

I have also seen the phenomenon you describe, with every plaque lighting 
up on the second screen.  Here's my theory.  I know from many results 
that there is cross-hybridization between plasmid vectors and lambda 
DNA.  I assume you are using a plasmid as a probe, and not isolating the 
insert.  In the first round screen, this background hybridization is 
uniformly distributed across all plaques, whereas your specific 
hybridization is limited to a few, thus you find them.  At the lower 
density of the second round screen, the amount of background hyb in each 
plaque is much greater - same probe divided among fewer plaques.  
However, if your probe is large and fairly well homologous to the insert 
of the right phage, you will see these as distinctly stronger signals.  
You can pretty much eliminate the problem by isolating the iinsert of the 
plasmid before you make the probe, or make it by PCR with flanking primers.
Hope this helps.

********************  HAVE GENES, WILL TRAVEL  ********************
Joe Bagshaw, Worcester Polytechnic Institute
jbagshaw at wpi.wpi.edu
Roadkill on the information superhighway.




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