Restriction Digestion Methods—Restriction Enzyme Digests

The unit definition of restriction enzyme activity is based on the amount of enzyme required to cut bacteriophage lambda DNA to completion in 1 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 that 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 that require more units per enzyme and longer incubation times for complete digestion). It is always best to use the manufacturer’s recommended assay conditions for restricted digestion. Some manufacturers have cloned enzymes that have very different requirements from other versions of the same enzyme, so check before using. Enzyme concentrations are always provided on the side of the enzyme tube. Restriction enzymes used in the lab are always stored at –20°;C (in a glycerol base), and should be kept as close to –20°;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 with 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.

Restriction Enzyme Buffers


Stock Solutions for Restriction Enzyme Buffers
Always check the enzyme manufacturer’s recommended buffer conditions before setting up digests. Prepare the 10X buffers by combining all ingredients except the BSA. Filter sterilize, then add the sterile BSA. Prepare 1-mL aliquots, and store at the recommended temperatures. Buffers containing 2-ME can be stored in the refrigerator for a few weeks. If the mercaptoethanol is undetectible in refrigerated buffer, adding 6 mL of the 12M 2-ME stock per mL will restore the buffer
- DTT (dithiothreitol), 100 mM stock: Prepare in dH₂O, filter sterilize and store at –20°;C.
- NaCl, 5 M stock: Prepare in dH₂O, filter sterilize, and store at room temperature.
- KCl, 1 M stock: Prepare in dH₂O, filter sterilize, and store at room temperature.
- Tris-HCl, pH 7.5-8.3, 1 M stock: Filter sterilize lab stock and store at room temperature.
- MgCl₂., 1 M stock: Filter sterilize lab stock and store at room temperature.
- 2-mercaptoethanol (2-ME), 12 M stock: Store in the refrigerator. Pipette under the hood.
- BSA (bovine serum albumin), 50 mg/mL stock: Purchase from BRL, store at –20°;C.

• Weigh out 0.25 g spermidine tetrahydrochloride.
• Add double distilled H₂O to bring volume to 10 mL.
• Filter, sterilize, and store at 4°;C.