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FAQ |  | Restriction Endonucleases FAQ
To see FAQs about a specific Restriction Endonucleases, see links from the specific product's page.
Q1: How can I search for a restriction enzyme by sequence, overhang or name?
Q2: How should I set up a restriction digest?
Q3: How should I stop my restriction digest?
Q4: How stable is a particular restriction enzyme?
Q5: I don't see any cleavage after my restriction digest. What factors can interfere with cleavage?
Q6: How can I generate a restriction enzyme site map for my sequence?
Q7: What information is available in the Restriction Enzyme Database (REBASE)?
Q8: Is extended digestion (incubation times > 1 hour) recommended?
Q9: Do degenerate recognition sites need to be palindromic?
Q1: How can I search for a restriction enzyme by sequence, overhang or name?
A1: Enzyme finder, a new tool available on our website in the sidebar under "Favorite Tools", can be used to search for restriction enzymes by name, sequence, overhang or type. NEB enzymes include enzyme properties icons and are displayed as links.
Q2: How should I set up a restriction digest?
A2: Most researchers follow the general rule that 10 units of restriction endonuclease is sufficient to overcome variability in DNA source, quantity and purity. Generally, 1 μl of enzyme is added to 1 μg 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.
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.
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 ther 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.
Q3: How should I stop my restriction digest?
A3: 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 catalog information for the particular enzyme(s) you are using. Phenol/chloroform extraction is another means of inactivating a restriction enzyme.
Q4: How stable is a particular restriction enzyme?
A4: All enzymes are assayed for activity every 3-6 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.
Q5: I don't see any cleavage after my restriction digest. What factors can interfere with cleavage?
A5: 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.
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.
Q6: How can I generate a restriction enzyme site map for my sequence?
A6: NEBcutter, a computer program for restriction enzyme site mapping is available on the NEB web site in the sidebar under "Favorite Tools". The program accepts sequences retrieved from a local file or GenBank. It aslo accepts an input sequence pasted into a designated field. When presented with a sequence, NEBcutter will find the largest open reading frames within the sequence and indicate those restriciton enzymes that could be used to excise the gene. It also locates the positions of all restriction enzymes that cut only once within the sequence chosen. NEBcutter is also fully aware of the information on the methylation sensitivity of restriction enzymes and alerts a user to overlapping methylation by dam, dcm, etc.
Q7: What information is available in the Restriction Enzyme Database (REBASE)?
A7: The Restriction Enzyme Database (REBASE), found in the sidebar under "Favorite Tools" on our web site, is a comprehensive database of information about restriction enzymes and related proteins. It contains published and unpublished references, recogntion and cleavage sites, isoschizomers, commercial availability, methylation sensitivity, crystal and sequence data. DNA methyltransferases, homing endonucleases, nicking enzymes, specificity subunits and control proteins are also included. Most recently, putative DNA methyltransferases and restriction enzymes, as predicted from analysis of genomic sequences, have been added.
Q8: Is extended digestion (incubation times > 1 hour) recommended?
A8: 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. This is contingent on how long a particular enzyme can survive (maintain activity) in a reaction. Some enzymes survive for long periods (> 16 hours) while others survive only an hour or less in a reaction. For each restriction enzyme, we report the minimum number of units (1.0, 0.5, 0.25 or 0.13) required to digest 1 µg of substrate DNA in 16 hours. Enzymes that require less than 1 unit can be used at lower concentrations for extended incubation times. Note that DNA substrates are digested at varying rates, the actual number of units required for a complete digestion will change from substrate to substrate. Check individual restriction enzyme information before extending reaction times, as those that exhibit star activity should be used under recommended conditions to inhibit noncanonical cleavage.
Q9: Do degenerate recognition sites need to be palindromic?
A9: Most restriction enzyme recognition sites are palindromic and include only specified base pairs (i.e., EcoRI recognizes GAATTC). However, some enzymes have degenerate sites, meaning that they contain one or more base pairs that are not specifically defined (i.e., BsrFI recognizes RCCGGY, where R= A or G and Y= C or T). For degenerate enzymes, any base represented by the single letter code may be present at either location in the recognition site for cleavage to occur. For example, BsrFI recognizes all of the following sequences: ACCGGC, ACCGGT, GCCGGC, GCCGGT.
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