flanking 5'-bases on BglI site within a primer

Allison allison at nospam.com
Thu Feb 9 14:45:25 EST 2006

Page 258 in the NEB 2005-2006 catalog has info on cleavage close to the 
end of fragments.  For Bgl II they got 100% cleavage with 3 bp extra. 
They suggest adding 4 extra nt onto the PCR primers (so 7 nt for Bgl II)

Did you clean up your PCR product before cutting?


Josmar wrote:
> Dear all,
> does anyone have experience how many flanking 5'-bases are needed on
> primers that were designed to introduce BglI sites into a pcr product?
> We have good evidence that our pcr-product with 5 additional bases is
> only very poorly cut, even overnight. We kind of rely on a
> quantitatively cut product because it contains a library. The NEB
> catalogue does not give a hint on this. I guess we will try 10
> additional bases next.
> Cheers
> Josmar

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