Primers incorporating restriction sites

Ashok Aiyar aiyar at
Wed Feb 17 08:41:01 EST 1999

On Wed, 17 Feb 1999 09:45:44 +0000,
    Jonathan Howarth (jrh2 at wrote:
>Can anyone tell me how well SacI and BamHI cut when 3 nucleotides from the
>end of a PCR fragment. These sites were incorporated into the 5' ends of
>the primers (+2 additional nucleotides ie. 5'NNGGATCC........3'). 
>I remember once seeing a table of such information for all the most common
>enzymes. Could anyone also tell me where I can find this table.

There's a table in the New England Biolabs catalog, and I think it
is also on their website (

If you find that either BamHI or SacI do not digest a significant 
fraction of your PCR product, you could try the following:

a) Phosphorylate the oligonucleotide primers prior to PCR.
b) Use either Vent/Pwo/Pfu for PCR, or "polish" the PCR product
   to make the ends blunt.
c) Self-ligate the product into a multimer.
d) Now digest with SacI and BamHI -- the majority of sites are 
   in the interior of the multimer and will be cut.

Several years ago I designed oligonucleotide primers that placed
HindIII and KpnI sites at the very ends of the primers.  I was 
unable to clone the product until I tried the strategy outlined above.

Ashok Aiyar, Ph.D.
McArdle Laboratory for Cancer Research
aiyar at

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