SOS!--PCR restriction cut

Bob Horton horto005 at maroon.tc.umn.edu
Tue Mar 19 09:08:56 EST 1996


ProZyme <info at prozyme.com> 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, 
1994.

---
Robert M. Horton
http://134.84.47.3                                       Have a :) day

"Scotty, try flushing the radioactive waste into the ventilation 
system!"



More information about the Methods mailing list