Frequently Asked Questions about REB and Restriction Enzymes:

1. How can I find the Restriction Enzymes which cleave a nucleotide sequence of my interest?
2. Is it possible for a Restriction enzyme to have varying recognition sequence?
3. What is the procedure for using a restriction enzyme?
4. How should I stop my restriction digest?
5. How stable is a particular restriction enzyme?
6. I don't see any cleavage after my restriction digest. What factors can interfere with cleavage?
7. Is extended digestion (incubation times > 1 hour) recommended?
8. Do degenerate recognition sites need to be palindromic?
9. How should restriction enzymes be stored?
10. How much enzyme should be used in a typical restriction enzyme reaction (also known as a digestion)?
11. Can too much restriction enzyme be used in a reaction?
12. How are restriction enzymes named?
13. How can restriction enzymes be diluted?

1. How can I find the Restriction Enzymes which cleave a nucleotide sequence of my interest?

REB provides you with a very efficient and multitask tool which has diverse functions. One among them is to find out the enzymes cleaving any given nucleotide sequence. Go to REB Tools and enter your sequence. The result will display all the commercially important enzymes which cleave your input sequence.

2. Is it possible for a Restriction enzyme to have varying recognition sequence?

Yes, it is possible that some enzymes may have more than one recognition sequence. Certain enzymes like AloI, AspCNI, BaeI, BfiI, I-BmoI, BpuJI, BslFI, BspCNI, CspCI, EcoPI, Hin4 and PstII produce variable cuts showing slight variation in their recognition sequences. Though the variation in the cuts has been detected the reason is yet to be found out.

3. What is the typical procedure for using a restriction enzyme?

A typical restriction enzyme digest can be set up as follows:
a) Determine volume of DNA to be digested.
b) Calculate how much enzyme is needed for complete digestion of the sample. In order that the final glycerol concentration is below 8%, consider the volume of enzyme to be, maximally, 10% of the final reaction volume (therefore the final glycerol concentration will be 5%).
c) The volume of 10X buffer should be 1/10 of the final reaction volume; the volume of 10X buffer will be no less than that determined for the enzyme;
d) Add together the volumes of DNA, 10X buffer, and enzyme calculated above. Make up the difference between this amount and the final volume (a standard reaction usually contains 20-50µl) with water. Mix gently and incubate at the appropriate temperature. Note: The appropriate temperature is most often 37°C, but there are exceptions.

For example:
Sterile, deionized water 16.3µl
RE 10x Buffer 2µl
Acetylated BSA, 10µg/µl 1µl
DNA, 1µg/µl 1µl
Restriction enzyme, 10u/µl 0.5µl
Final volume 20µl
Mix gently by pipeting, close the tube and centrifuge for a few seconds in a microcentrifuge. Incubate at the optimum temperature for 1-4 hours.

4. How should I stop my restriction digest?

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.

5. How stable is a particular restriction enzyme?

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. With growing research in restriction enzymes it has been found that most enzymes are very stable when stored at -20°C in the recommended storage buffer. Exposure to temperatures above -20°C should be minimized whenever possible.

6. I don't see any cleavage after my restriction digest. What factors can interfere with cleavage?

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.

7. Is extended digestion (incubation times > 1 hour) recommended?

Though incubation for one hour is recommended, 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.

8. Do degenerate recognition sites need to be palindromic?

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.

9. How should restriction enzymes be stored?

Restriction enzymes should be stored at -20°C in a non-cycling (not frost-free) freezer. The enzyme solution will not freeze at -20°C. Most enzymes can also be stored at -70°C for long term storage. However the enzyme solution will freeze at -70°C. As such, when the enzyme is used it will go through a freeze/thaw cycle. Restriction enzymes are sensitive to freeze/thaw cycles and enzyme activity will be lost with repeated freeze/thaws. It is recommended that if the enzyme is stored at -70°C it should be transferred to a -20°C freezer when the enzyme is first used. While it is being used, the enzyme should be kept on ice.

10. How much enzyme should be used in a typical restriction enzyme reaction (also known as a digestion)?

The amount of enzyme used is dependent on the amount, purity, and form of the DNA to be digested. The restriction enzyme unit is defined as the amount of enzyme required to produce a complete digest of 1µg of substrate DNA in 1 hour at the appropriate reaction temperature in a 50µl volume. In general, linear DNA is more readily digested than supercoiled DNA. For instance, using clean lambda phage DNA, 1-2 units of enzyme per 1µg of DNA is recommended. For supercoiled plasmid DNA, 5 units of enzyme for 1µg of DNA is appropriate.

11. Can too much restriction enzyme be used in a reaction?

Yes. In general, digestions will be inhibited by a glycerol content above 8%. In addition, some restriction enzymes are subject to star activity in high glycerol content. Since restriction enzymes are supplied in 50% glycerol, one should add no more than 1.6µl of enzyme per 10µl of total volume

12. How are restriction enzymes named?

The following order is used for designating restriction enzyme names:
a) a three letter abbreviation for the parent organism (for example, Bam for Bacillus amyloliquefaciens)
b) an additional letter or Arabic numeral, if necessary, to identify the strain or serotype (BamH)
c) and a Roman numeral to reflect the order of identification or characterization (BamH I)

13. How can restriction enzymes be diluted?

If the enzyme will be used within an hour it may be diluted in 1X reaction buffer supplemented with 0.5mg/ml acetylated BSA. If the diluted enzyme will be stored for longer periods it should be diluted in its storage buffer.