©CLONTECH	BIOL 327 Molecular Biology Lab Protocol #4 Restriction Enzyme Digestion of DNA
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Recombinant DNA technology was not really feasible until restriction endonucleases were discovered and isolated from bacteria. Restriction enzymes each have their own specific recognition site on double-stranded DNA, usually 6 to 8 bp in length and usually palindromic in sequence. These enzymes allow us to specifically cut long pieces of genomic DNA into manageable fragments and manipulate them. We will be performing several restriction digests during the course of our experiment. Some of these digest will be analytical, i.e. to determine the fragment length pattern of a particular piece of DNA. Other digest will be used to prepare the plasmid and genomic DNA for cloning. It is important that you understand the differences between these types of digests and when they are necessary.

Each restriction enzyme has a set of optimal reaction conditions, which are given on the information sheet and in the catalogues supplied by the manufacturer. The major variables in the reaction are the temperature of incubation and the composition of the reaction buffer. Most companies supply 10x concentrates of these buffers with the enzymes. These 10x buffers are usually stored at -20 oC. Some enzymes also require a non-specific protein. Usually bovine serum albumin (BSA) is used for this and is also supplied as a concentrated solution.

One unit of enzyme is usually defined as the amount of enzyme required to digest 1 ug of DNA to completion in 1 hour in the recommended buffer and temperature. In general, digestion for longer periods of time or with excess enzyme does not cause problems unless there is contamination with nucleases. Such contamination is minimal in commercial enzyme preparations. It is possible to minimize enzyme use (expensive reagent) by incubating for 2-3 hours with a small amount of enzyme.

When carrying out restriction enzyme digestions, prepare the reaction mixture up to the point here all reagents except the enzyme have been added and mixed. Take the enzyme from the freezer and immediately put it into ice. Use a fresh, sterile pipette tip every time you dispense enzyme. Contamination of an enzyme with DNA or another enzyme can be costly and time-consuming. Work as quickly as possible so that the enzyme is out of the freezer for as short a time as possible. If using the enzyme at your bench, keep it on ice at all times. Return enzyme to the freezer immediately after use!!

1. Many restriction enzymes are supplied by manufacturer in a concentrated form (~10-20U/ul). Often 1ul of many enzyme preparations is sufficient to digest 10ug of DNA in an hour. To remove small quantities of enzyme from the tube, touch the end of the pipette tip briefly to the surface of the fluid. In this way it is possible to remove as little as 0.1 ul of enzyme.
2. Restriction enzymes are stable when they are stored at -20oC (freezer) in buffer containing 50% glycerol.
3. Keep reaction volumes to a minimum by reducing the amount of water in the reaction as much as possible. However, make sure that the restriction enzyme contributes less than 1/10 of the final reaction volume (i.e. if reaction is 20ul then enzyme should be less than 2ul). This is due to the fact that some enzymes are inhibited by high levels of glycerol.
4. Often the amount of enzyme can be reduced if the digestion time is increased. This can result in considerable savings when large quantities of DNA are cleaved. Small aliquots can be removed during the course of the reaction and analyzed on a gel to monitor the progress of the digestion.
5. When digesting multiple DNA samples with the same enzyme, calculate the total amount of enzyme that is needed. Remove the correct amount of enzyme and mix it with the appropriate volume of 1 or 2x restriction buffer. Dispense aliquots of the enzyme/buffer mixture into the reaction tubes. Do this when screening mini-prep DNA samples.
6. When DNA is to be cleaved with two or more restriction enzymes, the digestions can be carried out simultaneously if both enzymes work in the same buffer. Alternatively, the enzyme that works in the buffer of lower ionic strength (salt concentration) should be used first. The appropriate amount of salt and the second enzyme can then be added and the incubation continued.
7. If the volume of the restriction enzyme reaction is too large to fit into the well of a gel, the DNA may be concentrated by the following simple procedure. After the reaction has been stopped by the addition of 1/20th volume 0.5M EDTA, add 1/10th volume of 3M sodium acetate and 2.5 volumes of cold ethanol. Set on ice for 15 minutes then centrifuge for 15 minutes in microfuge. Discard the supernatant which contains most of the protein. Dry the pellet briefly in the Speed-Vac and dissolve the DNA in the appropriate volume of TE.
8. Lots of useful information about restriction enzymes can be found in the New England Biolabs (NEB) catalogue. Highly recommended reading!