Traditional Cloning Workflow

Using PCR, restriction sites are added to both ends of a dsDNA, which is then digested by the corresponding REases. The cleaved DNA can then be ligated to a plasmid vector cleaved by the same or compatible REases with T4 DNA ligase. DNA fragments can also be moved from one vector into another by digesting with REases and ligating to compatible ends of the target vector.


The traditional cloning workflow has been used in molecular biology labs for decades. The protocol begins with insert DNA and a cloning vector. The insert DNA can be an individual fragment that has been amplified from a source DNA to annealed oligos or a restriction fragment from a vector. Cloning vectors are identifiable by the presence of a multiple cloning site or MCS. Restriction enzymes are then used to digest the insert and vector DNA to create compatible ends. Next, these parts are assembled using DNA ligase to form a covalently-sealed plasmid. Finally, the ligation reaction is transformed into competent e.coli and plated to identify successful transformants.

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