- Digest — the restriction enzyme recognizes a palindromic sequence and cuts both the donor DNA (around the gene of interest) and the plasmid vector at the same site, leaving complementary single-stranded "sticky ends."
- Ligate — the gene fragment's sticky ends base-pair with the vector's sticky ends; DNA ligase seals the phosphodiester backbone at both junctions, forming a closed recombinant plasmid.
- Transform — heat shock (~42°C pulse) transiently opens pores in the E. coli membrane, letting the recombinant plasmid enter the cell.
- Select — cells are plated on antibiotic medium. Only cells carrying the plasmid's resistance gene (and therefore the insert) survive and form colonies; untransformed cells die.
Enzyme site → sticky end
EcoRI G^AATTC → 5'-AATT overhang
BamHI G^GATCC → 5'-GATC overhang
HindIII A^AGCTT → 5'-AGCT overhang
This restriction–ligation–transformation–selection workflow is the classical route for building recombinant plasmids, still taught as the foundation of genetic engineering.