High-Fidelity (HF®) Restriction Endonucleases

For over 40 years, New England Biolabs has been developing innovative solutions for molecular biology applications. The respected leader in the field of restriction enzyme biology, NEB has developed a line of High-Fidelity (HF®) Restriction Enzymes. These engineered enzymes have the same specificity as the native enzyme, with the added benefit of reduced star activity, rapid digestion (5-15 minutes) and 100% activity in CutSmart® Buffer.  

Engineered with performance in mind, HF restriction enzymes are fully active under a broader range of conditions, minimizing off-target products, while offering flexibility in experimental design. 

Optimized performance for a wide range of conditions 

High Fidelity Restriction Enzymes have been engineered by exchanging functional amino acid residues and then screening for optimal mutants that perform under a wide range of conditions. Whether you are setting up digests for 5-15 minutes or overnight, or using varying amounts of enzymes, HF enzymes ensure the performance you need.

EcoRI-HF® (NEB #R3101) shows no star activity in overnight digests, even when used at higher concentrations. 50 μl reactions were set up using 1 μg of Lambda DNA, the indicated amount of enzyme and the recommended reaction buffer. Reactions were incubated overnight at 37°C. Marker M is the 1 kb DNA Ladder (NEB# N3232)



HF® is a registered trademark of New England Biolabs, Inc.
FastDigest® is a registered trademark of Fermentas.

FAQs for High-Fidelity (HF®) Restriction Endonucleases

Protocols for High-Fidelity (HF®) Restriction Endonucleases

    Publications related to High-Fidelity (HF®) Restriction Endonucleases

  1. 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

Benefits of High-Fidelity (HF®) Restriction Enzymes

  • One buffer convenience with no loss of performance
  • Reduced star activity eliminates unwanted cleavage
  • Time-Saver qualified for 5-15 minute digests, flexible enough to digest overnight.
  • Engineered for performance under a wide range of conditions 
  • Added flexibility without added cost


  • Cloning
  • Genotyping
  • Mutational Analysis
  • Mapping
  • Probe Preparation 
  • Sequencing 
  • Methylation Detection
  • Any application that requires high fidelity or flexible reaction setup

Legal Information

This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).

While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.

For more information about commercial rights, please contact NEB's Global Business Development team at

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

  1. ReduceStarActivity_video_thumb

    Reduce Star Activity with High-Fidelity Restriction Enzymes

    NEB has engineered HF® enzymes to eliminate star activity. Learn how, and what this means for your digests.

  2. Discovering New Restriction Enzymes

    Discovering New Restriction Enzymes

    Watch as Rick Morgan, Research Scientist in the Restriction Enzyme Division, describes his passion for discovering and characterizing restriction enzymes from nature.

  3. Restriction Enzyme Interaction with DNA

    The Interaction of Restriction Enzymes and DNA

    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.

  4. TooManyBands_video_thumb

    Restriction Enzyme Digest Problem: Too Many DNA Bands

    Are you finding unexpected bands in your digestion reaction? Here are some tips to help you determine the cause.

  5. whatisstaractivity_video_thumb

    What is Restriction Enzyme Star Activity?

    Learn what Star Activity is, why it is detrimental to accurate restriction enzyme digestion, and how NEB's HF enzymes are engineered to avoid it.

  6. DoubleDigestProtocol_video_thumb

    NEB Restriction Enzyme Double Digest Protocol

    Double digestions can save you time, and this video can offer tips for how to achieve the best results, no matter which of NEB's restriction enzymes you're using.

  7. dnasmear_video_thumb

    Restriction Enzyme Digestion Problem: DNA Smear on Agarose Gel

    Learn more about what causes this common problem, and how NEB's enzymes are QC'd to avoid DNA smearing.

  8. StandardREProtocol_video_thumb

    Standard Protocol for Restriction Enzyme Digests

    Let one of NEB's restriction enzyme experts help you improve your technique and avoid common mistakes in digest setup.

  9. cutsmart_video_thumb

    CutSmart Restriction Enzyme Buffer

    >210 of NEB's restriction enzymes are 100% active in a single buffer. Learn more about CutSmart® Buffer and why it matters to you.