Inactivation of restriction endonucleases is generally not necessary, but in some cases it might increase the transformation efficiency. If the insert also carries the restriction site that was used to linearize the vector 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.