There are several key factors to consider when setting up a restriction endonuclease digest. Using the proper amounts of DNA, enzyme and buffer components in the correct reaction volume will allow you to achieve optimal digestion without any star activity. By definition, 1 unit of restriction enzyme will completely digest 1 µg of substrate DNA in a 50 µl reaction in 60 minutes. This enzyme : DNA : reaction volume ratio can be used as a guide when designing reactions. However, most researchers follow the “typical” reaction conditions listed, where a 10-fold overdigestion is recommended to overcome variability in DNA source, quantity and purity. NEB offers the following tips to help you to achieve maximal success in your restriction endonuclease reactions.

A "Typical" Restriction Digest
Most researchers follow the general rules that 10 units of restriction enzyme is sufficient to overcome variability in DNA source, quantity and purity. Generally, 1 µl of enzyme is added to 1 ug of purified DNA in a final volume of 50 µl of the appropriate 1X NEBuffer followed by incubation for 1 hour at the recommended temperature. If an excess of enzyme is used, the length of incubation can often be decreased to save time. Alternatively, you can productively digest with fewer units of enzyme for up to 16 hours with many restriction enzymes.

Choosing the Right Enzyme
Obviously the DNA to be digested must contain a recognition sequence for the restriction enzymes chosen. Restriction enzymes with shorter recognition sequences cut DNA more frequently than those with longer recognition sequences. Assuming a 50% G-C content, a restriction enzyme with a 4-base recognition sequence will cleave, on average, every 44 (256) bases compared to every 46 (4096) bases for a restriction enzyme with a 6-base recognition sequence. Cleavage by a restriction enzyme produces either cohesive (having either a 5´ or 3´ single-stranded protrusion) or blunt-ended (no single-stranded protrusion) fragments. Cohesive fragments can be subsequently ligated to other restriction fragments if their single-stranded protrusions or "overhangs" are compatible. All blunt-ended fragments can be ligated to each other. See Compatible Cohesive Ends and Recleavable Blunt Ends.

Enzyme
Restriction enzymes should be kept on ice when they are not in the freezer. The enzyme should always be the last component added to a reaction (reaction components should be mixed prior to addition of enzyme). The number of units added to a reaction must be adjusted to the varying cleavage rates of DNA substrates. For example, supercoiled plasmids and agarose-embedded DNAs generally require more than 1 unit/µg to be cleaved completely. More information about cleavage of plasmid DNA and agarose-embedded DNA.

DNA
The preparation of DNA to be cleaved should be free of contaminants such as phenol, chloroform, alcohol, EDTA, detergents, or excessive salts, all of which can interfere with restriction enzyme activity. DNA methylation is also an important element of a restriction digest. More information about Effect of CpG Methylation on Restriction Enzyme Cleavage and Dam and Dcm Methylases of E.coli.

Reaction Buffer
New England Biolabs provides a color-coded 10X NEBuffer with each restriction enzyme to ensure optimal (100%) activity. The buffer should be used at 1X concentration in the reaction. Some restriction enzymes require bovine serum albumin (BSA) at a final concentration of 100 µg/ml for optimal activity. When required, BSA is supplied as a 10 mg/ml (100X) stock and should be added to the reaction mixture. Restriction enzymes that do not require BSA for optimal activity are not adversely affected if BSA is present in the reaction. More information about NEBuffers.

Reaction Volume
By definition, 1 unit of restriction enzyme will completely digest 1 µg of substrate DNA in a 50 µl reaction in 60 minutes. This enzyme : DNA : reaction volume ratio can be used as a guide when designing reactions. Smaller reaction volumes are more susceptible to pipetting errors. To keep glycerol concentration at less than 5% in a reaction, the restriction enzyme, which is supplied in 50% glycerol, should not exceed 10% of the total reaction volume.

Mixing
An extremely important, yet often overlooked, element of a successful restriction digest is mixing. The reaction must be thoroughly mixed to achieve complete digestion. We recommend gently pipetting the reaction mixture up and down or “flicking” the reaction tube. Follow with a quick (“touch”) spin-down in a microcentrifuge. Do not vortex the reaction.

Incubation Temperature
The recommended incubation temperature for most restriction enzymes is 37°C. Restriction enzymes isolated from thermophilic bacteria require higher incubation temperatures ranging from 50°C to 65°C. More information on the activity of thermophiles at 37°C.

Incubation Time
The unit definition of our restriction enzymes is based on a 1 hour incubation. Incubation time may be shortened if additional units of restriction enzyme are added to the reaction. Conversely, longer incubation times are often used to allow a reaction to proceed to completion with fewer units of enzyme. Refer to the specific information about enzyme survival times in a reaction.

Stopping a Reaction
If no further manipulations of the digested DNA are planned, the reaction can be terminated by adding a stop solution. At NEB we use the following stop solution: 50% Glycerol, 50 mM EDTA (pH 8.0), and 0.05% bromophenol blue (10 µl/50 µl reaction). If further manipulations of the digested DNA are required, heat inactivation (raising the temperature to 65 or 80°C for 20 minutes) is the simplest method of stopping a reaction. Since this method does not work for all restriction enzymes, refer to the heat inactivation chart. Phenol/chloroform extraction is another means of inactivating a restriction enzyme.

Storage
We recommend storage at –20°C for most restriction enzymes. For a few enzymes, storage at –70°C is recommended for periods longer than 30 days. Please refer to the enzyme's technical data sheet or catalog entry for storage information. 10X NEBuffers and concentrated BSA should also be stored at –20°C. BSA should not be mixed directly into NEBuffers and then frozen because the BSA may precipitate..

Stability
All enzymes are assayed for activity every 1-2 months; the most recent assay date is given on the label attached to each vial of enzyme. After thirty years of experience with restriction enzymes, we have found that most are very stable when stored at –20°C in the recommended storage buffer. Exposure to temperatures above –20°C should be minimized whenever possible.

Control Reactions
If you are having difficulty cleaving your DNA substrate, we recommend the following control reactions. Incubate experimental DNA without restriction enzyme (degradation of DNA indicates contamination in the DNA preparation or reaction buffer) and control DNA (DNA with multiple known sites for the enzyme, e.g. lambda or adenovirus-2 DNA) with restriction enzyme to more accurately judge whether or not the reaction went to completion. If the control DNA is cleaved and the experimental DNA resists cleavage, the two DNAs can be mixed to determine if an inhibitor is present in the experimental sample. If an inhibitor (often salt, EDTA or phenol) is present, the control DNA will not cut after mixing.