Frequently, a primer extension product must be further manipulated by cleavage with restriction enzymes. For convenience, restriction enzyme digestion can be performed directly in the primer extension mix without any purification of the DNA. This table summarizes the percent activity of restriction enzymes on the DNA in the primer extension mix described below.
5 units of restriction enzyme were incubated at the appropriate reaction temperature for 1 hour in a standard primer extension mix containing 1 µg of DNA and 1 unit of VentR® DNA Polymerase (NEB #M0254) in a 20 µl reaction volume of 1X ThermoPol Buffer (NEB #B9004) supplemented with dNTPs (200 µM final concentration). For some enzymes (BsiHKAI (NEB #R0570), BspEI (NEB #R0540), DraIII (NEB #R3150), SalI (NEB #R0138)), either NaCl (100 mM final concentration) or the appropriate 10X NEBuffer (1x final concentration) was added prior to digestion. Enzyme activity was analyzed by gel electrophoresis.
Note: The use of restriction enzymes under non-optimal conditions does increase the likelihood of star activity. To prevent star activity to optimize cleavage of restriction enzymes that do not perform well in a primer extension mix, the DNA can be purified by alcohol precipitation followed by redissolving in the appropriate restriction enzyme NEBuffer.
Please also check these additional information about:
Activity of Restriction Enzymes in a Taq or Phusion® PCR Mix
Chart Legend: Cleavage in extension mix with 5 units of enzyme:
+++ complete cleavage; ++ ~50% cleavage; + ~25% cleavage
VentR® is a registered trademark of New England Biolabs, Inc.
ThermoPol™ is a trademark of New England Biolabs, Inc.
Phusion® was developed by Finnzymes Oy, now a part of Thermo Fisher Scientific. This product is manufactured by New England Biolabs, Inc. Under agreement with, and under performance specifications of Thermo Fisher scientific.