Under such conditions, each cell accumulates several thousand copies of the plasmid so that one litre of bacterial culture easily yields a milligram of plasmid DNA. It has two selectable markers (tetracycline, tetr, and ampicillin, amp', resistance genes), and unique recognition sites for 12 different restriction enzymes (two unique sites, PstI and PvuI, are located within the amp' gene, and 4, e.g., BamHI, SalI, etc., are within tetr gene).

The presence of restriction sites within the markers tetr and ampr permits an easy selection for cells transformed with the recombinant pBR322. Insertion of the DNA fragment into the plasmid using restriction enzyme PstI or PvuI places the DNA insert within the gene amp'; this makes amp' nonfunctional. Bacterial cells containing such a recombinant pBR322 will be unable to grow in the presence of ampicillin, but will grow on tetracycline.

This eliminates nontransformed bacterial cells; the resulting bacterial colonies will posses either recombinant or unaltered pBR322. The colonies so obtained are then replicaplated on agar plates containing the other antibiotic (within the resistance gene for which the DNA insert is placed); all the colonies that develop on this plate will contain the unaltered pBR322.

Therefore, the antibiotic sensitive colonies are identified and recovered from the master plate; these colonies will have the recombinant pBR322. This entire process may take up to 2 days.