FAQ: Is it necessary to inactivate restriction enzymes after vector digestion?

No, unless the insert also carries the restriction site that was used to linearize the vector. You may still linearize the vector with such restriction enzymes, but it is necessary to heat inactivate the restriction enzyme before mixing the linearized vector with the insert in Gibson Assembly. If a heat-resistant restriction enzyme was used to linearize the vector, then vector should be purified by DNA columns, phenol-chloroform extraction or extracted from agarose gel after electrophoresis, before coming into contact with the insert.