High-Fidelity (HF®) Restriction Endonucleases

For over 45 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 rCutSmart™ and 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.

We are excited to announce that all reaction buffers are now BSA-free. NEB began switching our BSA-containing reaction buffers in April 2021 to buffers containing Recombinant Albumin (rAlbumin) for restriction enzymes and some DNA modifying enzymes. Find more details at

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.

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FAQs for High-Fidelity (HF®) Restriction Endonucleases
Protocols for High-Fidelity (HF®) Restriction Endonucleases
    Publications related to High-Fidelity (HF®) Restriction Endonucleases
    • Kamps-Hughes, N., Quimby, A., Zhu, Z., Johnson, E.A. (2013) Massively parallel characterization of restriction endonucleases Nucleic Acids Res ; 41(11), e119. 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

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email

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.