The Leader in the Discovery and Production of Restriction Enzymes
With over 40 years of offering restriction enzymes to the research community, NEB has earned the reputation of being a leader in enzyme technologies. Working continuously to be worthy of that distinction, NEB strives to develop enzymes of the highest purity and unparalleled performance.
NEB scientists continue to improve its portfolio of restriction enzymes, as well as explore their utility in new
technologies. As a result, NEB scientists continue to publish scientific papers and to be awarded grants in this area.
With the industry’s largest research and development group dedicated to restriction enzymes, we are proud to have
been there first: the first to commercialize a recombinant enzyme, the first to introduce a nicking enzyme, and the first
to supply a true restriction enzyme master mix. In addition, NEB has an ongoing history of innovation by engineering
restriction enzymes with altered specificities and improved performance. Through continued research in these areas, we
are committed to driving the innovations that allow us to offer maximum convenience and performance.
For details on NEB’s quality controls for restriction endonucleases, visit our Restriction Enzyme Quality page.
All of NEB's Restriction enzymes have transitioned to a new buffer system. Visit NEBCutSmart.com for further details.
- >215 restriction enzymes are 100% active in a single buffer – CutSmart™ Buffer.
- >195 restriction enzymes are Time-Saver qualified, meaning you can digest DNA in 5-15 minutes, or digest DNA safely overnight.
- Choose from >285 restriction enzymes, the largest selection commercially available.
- Choose a High-Fidelity (HF®) restriction enzyme, which has been engineered for reduced star activity, rapid digestion (5-15 minutes) and 100% activity in CutSmart Buffer. A vial of 6X Purple Loading Dye is included with most restriction enzymes.
- All of our restriction enzymes undergo stringent quality control testing, ensuring the highest levels of purity and lot-to-lot consistency.
Physical Purity Enzymes are evaluated by SDS-PAGE and silver-stained gels to ensure the highest levels of purity and the absence of contaminating proteins.
DNA Contamination Enzymes are screened by qPCR to ensure no contaminating genomic DNA is present. The specification for this assay is less than one E.coli genome per sample. Exonuclease Activity Using radioactively labelled DNA substrate and/or state-of-the-art capillary electrophoresis-based assays with fluorescently-labelled substrates, NEB is able to detect very low levels of exonuclease activity. Endonuclease Activity To ensure that there are no contaminating enzymes that could cause nickng or non-specific nuclease degradation, reagents are incubated with supercoiled plasmid DNA for 4 hours to demonstrate the absence of endonuclease contamination. Non-Specific DNase Activity Enzymes are incubated overnight with Lambda DNA to confirm that there is no additional non-specific nuclease activity present. Cloning QC (Ligation and Re-cutting) A DNA template is over-digested by the appropriate restriction enzyme and the percentage of DNA fragments ligated and re-cut are determined by agarose gel electrophoresis. Cloning QC (Blue-white Screening) A DNA plasmid is over-digested by the appropriate restriction enzyme and the linearized plasmid DNA is ligated and transformed into an E.coli strain with greater than 99% correct transformants, as determined by a blue-white screen.
Use Enzyme Finder to select restriction enzyme by name, sequence, overhang or type.
Restriction Endonucleases includes these subcategories:
FAQs for Restriction Endonucleases
Protocols for Restriction Endonucleases
- Double Digest Protocol using One RE-Mix and One Standard Restriction Enzyme
- Standard Digest Using RE-Mix®
- Double Digest Protocol using Two RE-Mix® Enzymes
- Digestion of Agarose-Embedded DNA
- Protocol for Direct Digestion of gDNA during droplet digital PCR (ddPCR)
- Protocol for Digestion Prior to droplet digital PCR (ddPCR)
- Optimizing Restriction Endonuclease Reactions
- Double Digest Protocol with Standard Restriction Enzymes
A Modern Day Gene Genie Sir Richard Roberts on Rebase
Restriction Endonucleases: Molecular Cloning and Beyond
Restriction Enzyme Cleavage: ‘single-site’ enzymes and ‘multi-site’ enzymes
Restriction enzymes are proteins used to fragment and clone DNA, but their biological function is to protect bacteria and archaea against viral infections.
Type II Restriction Enzymes: What You Need to Know | NEB
Read about Type II restriction enzymes and the distinguishing properties of the four principle subtypes.
Whole genome assembly from next generation sequencing data using restriction and nicking enzymes in optical mapping and proximity-based ligation strategies
High throughput sequencing methods have revolutionized genomic analysis by producing millions of sequence reads from an organism’s DNA at an ever decreasing cost.
The CutSmart™ Trifold provides information about the benefits of using CutSmart Buffer with NEB restriction enzymes.
NEB Restriction Enzyme Activity Poster
Find the optimal NEBuffer and approximate activity in the four standard NEBuffers and NEBuffer EcoRI for each enzyme, and learn the best conditions for double digests.
Restriction Endonucleases Technical Guide
The Restriction Enzyme Technical Guide provides product information and technical reference charts on the wide range of restriction enzymes available from NEB.
- Crystal Structure of the 8 bp-Specific Restriction Enzyme SwaI (2015)
- DNA Sequences and Maps Tool
- Alphabetized List of Recognition Specificities
- Average Fragment Size Generated By Endonuclease Cleavage
- Compatible Cohesive Ends and Generation of New Restriction Sites
- Cross Index of Recognition Sequences
- Dam-Dcm and CpG Methylation
- Enzymes with Multiple Recognition Sequences
- Enzymes with Nonpalindromic Sequences
- Frequencies of Restriction Sites
- Interrupted Palindromes
- Recleavable Blunt Ends
- Recleavable Filled-in 5' Overhangs
- Time-Saver™ Qualified Enzymes
- Why Choose Recombinant Enzymes?
- Restriction Enzyme Troubleshooting Guide
- Activity at 37°C for Restriction Enzymes with Alternate Incubation Temperatures
- Activity of Restriction Enzymes in PCR Buffers
- Alteration of Apparent Recognition Specificities Using Methylases
- Cleavage Close to the End of DNA Fragments
- Cleavage Of Supercoiled DNA
- Dam and Dcm Methylases of E. coli
- Digestion of Agarose-Embedded DNA: Info for Specific Enzymes
- Double Digests
- Effects of CpG Methylation on Restriction Enzyme Cleavage
- Heat Inactivation
- Megabase Mapping
- NEBuffer Activity/Performance Chart with Restriction Enzymes
- Optimizing Restriction Endonuclease Reactions
- Reduced Star Activities of HF® Enzymes
- Restriction Endonucleases - Survival in a Reaction
- Restriction Enzyme Diluent Buffer Compatibility
- Restriction Enzyme Tips
- Restriction Enzymes for Droplet Digital PCR (ddPCR)
- Restriction Enzymes requiring multi-sites for efficient cleavage
- Restriction of Foreign DNA by E. coli K-12
- Site Preferences
- Star Activity
- Traditional Cloning Quick Guide
Other Tools & Resources
- Shah, S., Sanchez, J., Stewart, A., et al. 2015. Probing the Run-On Oligomer of Activated SgrAI Bound to DNA PLoS One. 10(4), PubMedID: 25880668, DOI: 10.1371/journal.pone.0124783.
- Loenen, W.A., Raleigh, E.A. 2014. The other face of restriction: modification-dependent enzymes. Nucleic Acids Res. 42, PubMedID: 23990325, DOI:
- Kamps-Hughes, N., Quimby, A., Zhu, Z., Johnson, E.A. 2013. Massively parallel characterization of restriction endonucleases Scopus. 41(11), PubMedID: 23605040, DOI: 10.1093/nar/gkt257
- Fu YB, Peterson G. W., Dong Y 2016. Increasing Genome Sampling and Improving SNP Genotyping for Genotyping-by-Sequencing with New Combinations of Restriction Enzymes G3. 6:4, PubMedID: 26818077, DOI:
Publications related to Restriction Endonucleases
These days, restriction enzymes are being used in many more applications other than cloning. Learn more in this episode of NEB TV.
Type II restriction enzymes are most commonly used for molecular biology applications, as they recognize stereotypical sequences and produce a predictable cleavage pattern. Learn more about how Type II REs work.
Type I restriction enzymes are a group of endonucleases that recognize a bipartite sequence, but do not produce a predictable cleavage pattern. Learn more about how Type I REs work.
Type III restriction enzymes are a group of endonucleases that recognize a non-pallindromic sequence, comprising two inversely oriented sites. Learn more about these poorly understood enzymes.
Watch as Geoff Wilson, Restriction Enzyme Division Head, describes the interaction of restriction enzymes and substrate DNA using computer models generated from x-ray crystallography data.
Watch as Rick Morgan, Research Scientist in the Restriction Enzyme Division, describes his passion for discovering and characterizing restriction enzymes from nature.
Chromatin conformation capture (3C) techniques allow study of the spatial organization of eukaryotic chromosomes in a 3D context.
Droplet digital PCR is a method for accurately quantitating copies of DNA or RNA in a sample. Each PCR reaction is separated into thousands or millions of droplets for analysis. Learn more about droplet digital PCR.
Type IIS restriction enzymes have both recognition and binding sites, but cut downstream of the recognition site, creating 4-base overhangs ideal for re-assembly. View a list of TypeIIS enzymes.
Isothermal amplification generates many copies of a target sequence in a short period of time, at a constant temperature. Learn more about isothermal amplification.
Optical mapping is a method that allows production of restriction maps of whole chromosomes or genomes. Learn more about optical mapping.