Quick Tips - Should I clean up my PCR before T7 endonuclease digestion?

A lot of customers ask us "Do I need to clean up my PCR reaction before T7 endonuclease digestion?"

This depends on which polymerase you are using. Many polymerase buffers have components which can inhibit the T7 endonuclease reaction. We have tested several of our polymerases listed here in their unique buffers.  When using one of them, we find that you're able to add up to four microliters to a 50 micro liter reaction without a problem.  A full protocol is available on the NEB website. 

• Q5 High-Fidelity DNA polymerase with Q5 reaction buffer

• OneTaq with Standard buffer

• Taq DNA polymerase with ThermoPol buffer

For any other polymerase we would recommend cleaning up prior to digestion. For cleanup we would recommend using one of our monarch PCR DNA cleanup kits. To make things easier, we have available the EnGen Mutation Detection Kit which includes the Q5 2x master mix and T7 Endonuclease enzyme. This kit was designed to avoid the PCR cleanup step.

If you have any more questions you can contact us at info@neb.com.