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June 25, 1990
C. Helms
The unit definition of restriction enzyme activity is based on the amount of enzyme required to cut bacteriophage lambda DNA to completion in one hour's time. 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 ard 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.
The lab has prepared enzyme buffer stocks. Check the requirements of the enzyme and use the appropriate buffer. Nearly all the manufacturers of enzyme now supply buffers with the enzyme, and these can be found in the enzyme freezer. Never make up a digest having the enzyme as more than 1/10 the final volume; the glycerol will inhibit at higher concentrations. Always set up a control digest when using an enzyme. Use a substrate DNA which has known sites for the enzyme so that you can compare the control result to the expected pattern of fragments on an agarose gel. If preparing double enzyme digests, check the salt content of the buffers and use the enzyme with the lower salt requirements first, then adjust the reaction tube's salt concentration and digest with the second enzyme. Impurities present in some human DNA preparations may inhibit restriction digestion. If you cannot obtain complete digestion after adding additional enzyme, set up another digest and add spermidine to a final concentration in the digest of 2 mM.
References:
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.