Frequently, a PCR product must be further manipulated by cleavage with restriction enzymes. For convenience, restriction enzyme digestion can be performed directly in the PCR mix without any purification of the DNA. This table summarizes the percent activity of restriction enzymes on the DNA in the Taq, Phusion® or Q5® PCR mixes described below.
In these reactions, 5 units of restriction enzyme were incubated at the appropriate reaction temperature for 1 hour in a PCR mix containing 1 µg of DNA and 1 unit of DNA Polymerase in a 50 µl reaction volume with a final buffer concentration of 1X, and supplemented with dNTPs (200 µM final concentration). Enzyme activity was analyzed by gel electrophoresis.
Notes: The polymerase is still active and can alter the ends of DNA fragments after they
have been cleaved, affecting subsequent ligation. Primers containing the recognition site
of the restriction enzyme can act as competitive inhibitors in the cleavage reaction. The
use of restriction enzymes under non-optimal conditions does increase the likelihood of star
activity. If any problems are encountered, the DNA should be purified using a commercial
spin column (we recommend the Monarch PCR & DNA Cleanup Kit, NEB# T1030) or by phenol/chloroform followed by alcohol precipitation.
*It has been shown that the addition of 1x restriction enzyme buffer may help to improve the ability of some enzymes to cleave.
Cleavage in extension mix with 5 units of enzyme:
+++ complete cleavage; ++ ~50% cleavage; + ~25% cleavage;
– no activity