Restriction Enzyme Digests
JoVE Science Education Database. Basic Methods in Cellular and Molecular Biology. Restriction Enzyme Digests. Journal of Visualized Experiments, Cambridge, MA, doi: 10.3791/5070 (2014).
Restriction enzymes or endonucleases recognize and cut DNA at a specific sequence. These enzymes occur naturally in bacteria as a defense against bacteriophages – viruses that infect bacteria. Bacterial restriction enzymes cut the invading bacteriophage DNA while leaving the bacterial genomic DNA unharmed due to addition of methyl groups.
This video explains the basic principles of restriction enzymes including: how restriction enzymes are named and the types of recognition sites and overhangs that exist. Also provided is a step-by-step generalized procedure for how to set up a restriction digest including the necessary components, the order in which the mixture should be assembled, and the typical incubation temperature and time. The importance of inactivating restriction enzymes to prevent star activity is mentioned. Tips for performing multiple enzymes digests and using controls in digestion reactions are also provided.