基于限制性内切酶molecular cloningis the "classic" cloning method and is still one of the most popular techniques. In principle, the plasmid assembly involves these following steps:
Restriction digest:Closed circular vector plasmid and gene of interest fragment are cleaved with one or more限制性内切酶.
Ligation:DNA fragments with compatible overhangs are ligated into a linearizedvector plasmid.
Higher success rates are achieved when using DNA fragments with 5' or 3' sticky/overhang ends rather than assembling DNA fragments with blunt ends.