SOS!--PCR restriction cut

Bob Horton horto005 at
Tue Mar 19 09:08:56 EST 1996

ProZyme <info at> wrote:

> ...You don't say how you purify your PCR product before
> cutting it; I think this is an important step.  I use the Geneclean 
> kit from Bio101 and I follow the "Double Geneclean" procedure. 

Ditto. The problem may not be that the enzyme doesn't cut, but that the
sticky end gets filled in by Taq and residual dNTPs. Both EcoRI and 
BamHI leave 5' overhangs, and are susceptible to this. See:

Bennett, B.L. and Molenaar, A.J. Cloning of PCR products can be 
inhibited by Taq polymerase carryover. BioTechniques 16 (1): 32-33, 

Robert M. Horton                                       Have a :) day

"Scotty, try flushing the radioactive waste into the ventilation 

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