Blunt-end cloning

John Donald jdonald at deakin.edu.au
Fri Jun 14 15:05:20 EST 1996


I have been using Stratagenes pCRscript blunt end cloning kit with 
limited success.  I often get many blue colonies and as a rule the 
white colonies are not recombinants.  The PCR products are polished 
prior to cloning and are added to the cloning reaction at 40-100:1 
insert to vector.  It seems to me the Srf1 enzyme is not cutting the 
plasmid that efficiently and therefore the vector is somehow 
preferentially ligating on itself.  I have read that blunt end 
cutters can delete nucleotides.  Is this true.  Any advice would be 
appreciated.



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