Restriction Enzyme Digest Protocol: Cutting Close to DNA End

Yvette Luyton:

For optimal cleavage of a restriction site occurring near the end of a DNA molecule, most often you're going to need some number of nucleotides between the restriction site and the end of your DNA molecule. For example, if you're designing a primer containing a restriction site, we recommend as a rule of thumb adding six nucleotides between the recognition site and the five prime end of the primer. This provides plenty of DNA for the enzyme to bind allowing for efficient cleavage. Make sure when adding those six bases that they're not a palindromic sequence and that they do not form primer dimers.

We have experimentally tested virtually all of the restriction enzymes commercially available from NEB to determine the minimum number of nucleotides required for optimal cutting at the end of the DNA molecule. For additional information please see the Cleavage Close to the End of DNA Fragments chart available on the NEB website.