Preparation of DNA for traditional cloning methods is dependent upon restriction enzyme digestion to generate compatible ends capable of being ligated together. The DNA to be cloned can vary widely, from genomic DNA extracted from a pure bacterial culture or a mixed population, to a previously cloned gene that needs to be moved from one vector to another (subcloning). Restriction enzymes can also be used to generate compatible ends on PCR products. In all cases, one or more restriction enzymes are used to digest the DNA resulting in either non-directional or directional insertion into the compatible plasmid.
Genomic DNA, regardless of the source, is typically digested with restriction enzymes that recognize 6-8 consecutive bases, as these recognition sites occur less frequently in the genome than 4-base sites, and result in larger DNA fragments. The desired insert size for the clone library determines which enzymes are selected, as well as the digestion conditions. Most often, a serial dilution of the selected restriction enzyme(s) is used to digest the starting material and the desired insert size range is isolated by electrophoresis followed by gel extraction of the DNA. This method of preparation provides DNA fragments of the desired size with ends compatible to the selected vector DNA.