Restriction Digestion Table of Contents
- Restriction Digestion Introduction
- Restriction Enzyme Digestion Protocol
- Restriction Enzyme Digestion Protocols
- Restriction Digestion Videos
- Restriction Enzyme Digestion Forum
- Restriction Enzyme Digestion Help
- Restriction Digestion Troubleshooting
©2014 Molecular Station. Updated February 2014.
Information on how to become an expert on Restriction Enzymes and digesting DNA !
Background on Restriction Enzymes
Restriction enzymes (or restriction endonucleases) are enzymes that cut double-stranded DNA by making two breaks, one through each of the phosphate backbones of the double helix. The enzyme does this without damaging the bases of the DNA. Although the enzyme breaks the DNA, the chemical bonds can be reformed by other enzymes known as DNA ligases. Therefore, restriction fragments of DNA from different chromosomes or genes can be ligated together, providing their ends are complementary.
Usefulness of Restriction Enzymes in Molecular Biology
The fact that DNA could be manipulated and different DNA pieces could now be spliced together, opened up new areas of research never before imagined. Many of the methods in molecular biology today rely on restriction enzymes. "Restriction" is derived from the fact that these enzymes were initally discovered in E. coli which appeared to be restricting the infection of specific bacteriophages ("viruses" of bacteria). It is believed restriction enzymes are a host defense mechanism evolved by bacteria and other organisms to resist viral infections, and to help with the elimination of viral sequences.
In 1978, the Nobel Prize for Medicine was awarded to Werner Arber, Daniel Nathans and Hamilton Smith for their discovery of restriction endonucleases, which lead to the development of recombinant DNA technologies. The first practical use of restriction enzymes in science and medicine was the manipulation of E. coli bacteria to express recombinant human insulin for diabetics.
Handling and Using Restriction Enzymes
A unit of restriction enzyme activity is defined by the amount of restrition enzyme required to cut 1 microgram of bacteriophage lambda DNA to completion in a time of 1 hour.
Assays developed by the manufacturer of the enzymes are most likely done using highly purified DNA (i. e. plasmids or lambda phage DNA) as substrate, and assay conditions that produce the best results with their particular preparation. Often the laboratory conditions are not as ideal, and a slight excess of enzyme or a longer incubation period is used to help ensure complete digestion.
There are many 'universal' enzyme buffers which will work with a variety of enzymes, but often they do not meet the most efficient requirements for any one enzyme. Because the universal buffers are not as efficient, we do not recommend their routine use in the lab (especially for complex genomic DNAs; digests of complex genomic DNAs are usually at high DNA concentrations which will inhibit the enzyme; also, the relatively crude DNA preps may contain inhibitors which requires more units enzyme and longer incubation times for complete digestion). It is always best to use the manufacturer's recommended assay conditions for restricition digestion. Some manufacturers have cloned enzymes which have very different requirements from other versions of the same enzyme, so check before using. Enzyme concentrations are always given on the side of the enzyme tube. Restriction enzymes used in the lab are always stored at -20 degrees C (in a glycerol base), and should be kept as close to -20 degrees C as possible to extend the life of the enzyme. When setting up digests, bring the reaction tube to the enzyme freezer in an ice bucket; remove the enzyme tube from the freezer and keep the tube on ice while working with the enzyme. Immediately return the tube to the freezer when finished. Use the pipetmen designated for restriction enzymes only and always use a fresh pipetman tip when removing enzyme from the stock tube.
Tips on Restriction Enzyme Usage
- All manufacturer's of restriction enzymes supply specific buffers with the enzymes, and these should be stored in the -20°C freezer. Restriction Enzymes should also be stored at -20°C.
- Never make up restriction digests with the restriction enzyme composing more than 1/10 of the final volume. This is due to the fact that the restriction enzyme proteins are stored in glycerol. At concentrations above 10%, glycerol not only inhibits the digestion but also can cause star activity - leading to aberrant, non-specific cuts of the DNA.
- Set up a control digest when using restriction enzymes. This helps you know if the enzyme is working properly and you set the reaction up well. Use a plasmid DNA which generates known fragmentation patterns.
- When preparing double enzyme digests, determine the salt content of the buffers. Conduct restriction digestion using the enzyme with the lower salt requirements FIRST, then use the second enzyme by adjusting the reaction tube's buffer to the second optimal salt concentration.
- DNA preparations may have impurities which can inhibit restriction enzyme digestion activity. Some DNA isolation kits even use high EDTA buffers to elute the DNA. This is ok for DNA storage, but high concentrations of EDTA can inhibit restriction digestion. PCR purify your DNA and elute with TE or water if your enzyme activity is inhibited but your DNA looks ok. You could also add Mg2+ to the reaction and see if that helps also.
- If you cannot obtain complete restriction digestion of your DNA after adding extra enzyme, set up a new digest and add spermidine to a final concentration of 2 mM.
Restriction Enzyme Digestion of DNA
Analytical versus Preparative Restriction Enzyme Digestions
Usually DNA digestions are conducted in 25 - 50 ul (microliters), when you just want to check or analyze your DNA. These are called analytical restriction enzyme digestions.
One can digest 0.25 to 2 ug (micrograms) for analytical preparations. This is because on an agarose minigel (30ml), one can visualize approximately .05ug (50 ng, nanograms) per band. Visualization of a band depends on the length of the DNA and the amount of DNA present. In general, the longer the DNA present in the band, the easier is will be to see.
DNA digestions of many micrograms and higher volumes from 50 - 500 ul (microliters) or more are preparative, meaning that you are preparing DNA fragments for subsequent purification and cloning.
Restriction Digestion of DNA protocol
Recipe for Restriction Enzyme Digestion:
10X Restriction Enzyme Buffer
(many micrograms of DNA)
|"X" ul DNA 20 or more ul|
dH2O(millipore or autoclaved)
|86 - X ul|
You might add Mg2+ to the digestion reaction (in the form of MgCl) if you use a large volume of DNA. You should do this especially if the buffer you eluted your DNA in contained EDTA. This is because if "X" is large, ie > 20ul volume, you may need to add Mg. EDTA binds to 4 moles of Mg for every mole of EDTA. Thus, DNA in a 1 mM (milimolar) solution of EDTA will bind 4mM of Mg. A good rule of thumb is to add 1 1ul of 10mM MgCl to every 20ul of DNA.
10X Restriction Enzyme Buffer
|DNA||"X" ul DNA 0.25 to 10 ul|
dH2O(millipore or autoclaved)
|43 - X ul|
Incubate at 37°C for 1 - 3 hours.
1 unit is enough enzyme to digest1 ug DNA at least in 1 hour. Digesting for 3 hours you could digest higher amounts of DNA. As you are adding 10-20 Units of enzyme, you could even digest 10ug of DNA in 1 hour ! Enzymes are expensive don't waste them needlessly!
Notes on Restriction Enzyme Usage :
- Keep enzymes cold.
- Always keep restriction enzymes on ice !
- Minimize the time you have restriction enzymes on ice. You can do this easily by purchasing a cold box (which is basically a block of metal you keep your enzymes in)
Sambrook, J., Fritsch, E.F., and T. Maniatis.(1989) Molecular Cloning, A Laboratory Manual. Second edition. Cold Spring Harbor Laboratory Press. pp 5.3- 5.32.
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