Il DNA purificato è poi trattato con un'enzima di restrizione in modo da generare frammenti le cui estremità siano coesive con quelle dsel vettore. Se necessario, il sito di restrizione desiderato può essere aggiunto durante la PCR.
=== Transformation ===
Following the'' in vitro'' manipulation of the DNA, the product is typically introduced into ''E. coli'' in a process called transformation. For transformation to happen, bacteria must be in a state of competence. Some bacteria show natural competence, in others this state must be induced by laboratory procedures. Typically, the cells are incubated in a solution containing divalent cations (often calcium chloride) under cold conditions, before being exposed to a heat shock.
The surface of bacteria such as ''E. coli'' is negatively charged due to phospholipids and lipopolysaccharides on its cell surface, and the DNA is negatively charged as well. One function of the divalent cations therefore would be to shield the charges by coordinating the phosphate groups and other negative charges, thereby allowing a DNA molecule to adhere to the cell surface. It is suggested that exposing the cells to divalent cations in cold condition may also change or weaken the cell surface structure of the cells, making it more permeable to DNA. The heat-pulse is thought to create a thermal imbalance on either side of the cell membrane, which forces the DNA to enter the cells through either cell pores or the damaged cell wall.
Electroporation is another transformation method. In this method the cells are briefly shocked with an electric field of 10-20 kV/cm which is thought to create holes in the cell membrane through which the plasmid DNA may enter. After the electric shock the holes are rapidly closed by the cell's membrane-repair mechanisms.
