After running the gels for 12 hours, they are stained with ethidium bromide and viewed under UV light for image capturing. Before running the gel, however, the DNA needs to be treated and prepared. One is the control DNA, and the other is incubated with a restriction endonuclease. Restriction endonucleases, or restriction enzymes, are found naturally in bacteria and are responsible for recognizing a certain sequence of DNA and cutting at a site on that sequence. NotI. NotI has unique properties that make it ideal for BAC DNA insert sizing.
Some restriction enzymes recognize sequences that are short and therefore common in the genome.
NotI, however, has one of the largest recognition sites, with 8 base pairs. These two characteristics result in rare cutting along the human genome. For BAC inserts, whose average size is around 100kb, probability favors no cutting at all. However, the NotI fragment should always be smaller than the uncut control. The DNA isolated from the BACs is still enclosed in the cloning vector, which is engineered to contain a NotI recognition sequence.