High-Fidelity (HF®) Restriction Endonucleases

Restriction Endonucleases
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  • 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.

    1. 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.

    2. 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.

    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