The first step in producing a recombinant plasmid is to identify which gene or genes to introduce into a preexisting plasmid. In addition to the gene(s) of interest, an indicator gene is chosen to differentiate recombinant cells from non-recombinant cells in later steps of the process. The most common indicator gene is an antibiotic resistance gene not native to the host species so that only the recombinant cells will have the antibiotic resistance gene conferred by the plasmid.
Restriction enzymes cut DNA only at specific sequences. Some produce so-called "sticky" ends, in which one strand is a few base pairs longer than the other, while others produce blunt ends, in which both strands are cut at the same location. Restriction maps showing all known restriction enzyme cut sites are available for many plasmids. Using these maps, scientists choose compatible restriction sites in the plasmid and on either side of the genes of interest in order to cut the genes out of their native DNA and make openings for them in the plasmid.
Restriction enzyme digestion is the process of incubating the DNA to be cut with the appropriate restriction enzymes. Because the desired DNA is now mixed in with scraps of other DNA, scientists use gel electrophoresis to separate the fragments by size and isolate the desired DNA. This is followed by ligation, in which the ends of the selected genes are joined with the ends of the plasmid. The recombinant plasmid now contains the genes of interest and the antibiotic resistance gene.
Amplification is the mass copying of genetic material. In the case of a recombinant plasmid, the plasmid is introduced into bacterial cells. Not all cells will successfully take in a plasmid so the cells are then grown on media containing an antibiotic that is lethal to non-recombinant bacteria. Because the recombinant cells have the antibiotic resistance gene conferred by the plasmid, only they will survive and multiply. The cells are then lysed, or broken, and the DNA is extracted and purified.