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  • EagI-HF®

    High Fidelity Enzymes

    Product Update: A free vial of Gel Loading Dye, Purple (6x)  is now included with all HF restriction enzymes.

    cloned at neb recombinant engineered timesaver 5min incubation temp heat inactivation cpg blue white
    Catalog #SizeConcentrationPriceQtyAdd to Cart
    R3505S500 units20,000 units/ml$62.00Add to Cart
    R3505L2,500 units20,000 units/ml$249.00Add to Cart
    R3505M2,500 units100,000 units/ml$249.00Add to Cart
    High-Fidelity (HF®) Restriction Endonucleases,
    Restriction Endonucleases: C-G,
    Time-Saver™ Qualified Restriction Enzymes
    Restriction Enzyme Digestion


    High Fidelity (HF®) Restriction Enzymes have 100% activity in CutSmart™ Buffer; single-buffer simplicity means more straightforward and streamlined sample processing. HF enzymes also exhibit dramatically reduced star activity. HF enzymes are all Time-Saver™ qualified and can therefore cut substrate DNA in 5-15 with the flexibility to digest overnight without degradation to DNA. 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.

    NEB extensively performs quality controls on all standard and high-fidelity (HF®) restriction enzymes. Examples of nuclease contamination studies for some of our HF restriction enzymes are shown below.

    Restriction Enzyme Competitor Study: Nuclease Contamination

    EcoRI, NotI, and BamHI from multiple suppliers were tested in reactions containing a fluorescent labeled single stranded, double stranded blunt, 3’overhang or 5’ overhang containing oligonucleotides. The percent degradation is determined by capillary electrophoresis and peak analysis. The resolution is at the single nucleotide level.

    Product Source

    An E. coli strain that carries the cloned and modified EagI gene from Enterobacter agglomerans (R. Morgan)

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    CutSmart® Buffer-2010X
    Gel Loading Dye, Purple (6X)256X

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme required to digest 1 µg of pXba DNA in 1 hour at 37°C in a total reaction volume of 50 µl.

    Reaction Conditions

    1X CutSmart® Buffer
    Incubate at 37°C

    1X CutSmart® Buffer:
    50 mM Potassium Acetate
    20 mM Tris-acetate
    10 mM Magnesium Acetate
    100 μg/ml BSA
    pH 7.9 @ 25°C

    Activity in NEBuffers

    NEBuffer 1.1: 25%
    NEBuffer 2.1: 100%
    NEBuffer 3.1: 100%
    CutSmart® Buffer: 100%

    Diluent Compatibility

    Storage Temperature


    Storage Conditions

    10 mM Tris-HCl
    500 mM NaCl
    1 mM DTT
    0.1 mM EDTA
    200 μg/ml BSA
    50% Glycerol
    pH 7.5 @ 25°C

    Heat Inactivation

    65°C for 20 min

    Methylation Sensitivity

    dam methylation: Not Sensitive
    dcm methylation: Not Sensitive
    CpG Methylation: Blocked


    1. EagI-HF has the same specificity as EagI (NEB #R0505), but it has been engineered for reduced star activity.
    2. Based on the stability of the enzyme in the reaction, incubations longer than 1 hr will not result in improved digestion, unless additional enzyme is added. Please refer to Restriction endonuclease survival in a reaction for more information regarding this topic.


    1. Is there any difference in the methylation sensitivity between EagI-HF and EagI?
    2. What is the difference between EagI-HF and EagI?
    3. How does the level of star activity of EagI-HF compare to EagI?
    4. When should I choose the HF version of an enzyme?
    5. When is star activity a concern?
    6. Why does the HF version of the enzyme have a different recommended buffer than the wild type enzyme?
    7. When should I choose the High Fidelity (HF) version of the enzyme?
    8. Can the change in buffer preference of the HF enzyme be advantageous?
    9. Will the HF enzyme produce elevated star activity when used in a buffer other than the one recommended?
    10. What does it mean to be Time-Saver™ qualified?
    11. How is the improvement in fidelity of HF restriction endonucleases quantitated?
    12. What is the Fidelity Index (FI)?
    13. Is there a difference in cutting close to the ends between EagI-HF and EagI?
    14. What does HF® refer to following the name of a restriction enzyme?
    15. What effect does BSA have on the performance of NEB’s restriction enzymes when included in the new buffers?
    16. Do I have to set-up digests with Time-Saver™ qualified enzymes for 5-15 minutes? Can I digest longer?
    17. How can I access the old NEBuffer Activity Chart?
    18. Is Gel Loading Dye, Purple (6X) or Gel Loading Dye, Purple (6X), no SDS compatible with other DNA binding dyes such as SYBR® and GelRed™ during gel electrophoresis?
    19. Can Gel Loading Dye, Purple 6X (B7024) be stored in cold temperatures?
    20. Why is my Restriction Enzyme not cutting DNA?
    21. Why do I see a DNA smear on an agarose gel after a restriction digest?
    22. Why do I see additional DNA bands on my gel after a restriction digest?
    23. How many nucleotides do I have to add adjacent to the RE recognition site in order to get efficient cutting?


    1. Optimizing Restriction Endonuclease Reactions
    2. Double Digest Protocol with Standard Restriction Enzymes
    3. Time-Saver Protocol for Restriction Enzyme Digests

    Selection Charts

    Usage Guidelines & Tips

    Troubleshooting Guides

    Interactive Tools

    Quality Control

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page. Further information regarding NEB product quality can be found here.
    • Blue-White Screening (Terminal Integrity):
      A sample of DNA vector linearized with a 10-fold excess of a restriction endonuclease, religated and transformed into an E. coli strain expressing the LacZ beta fragment gene results in less than 1% white colonies.
    • Endonuclease Activity (Nicking):
      The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
    • Exonuclease Activity (Radioactivity Release):
      The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
    • Ligation and Recutting (Terminal Integrity):
      After an over-digestion of DNA with a restriction endonuclease the percentage of the DNA fragments ligated with T4 DNA ligase and the percentage that can be recut are determined by agarose gel electrophoresis.
    • Non-Specific DNase Activity (16 hour):
      The product is tested for non-specific nuclease degradation in a reaction containing a DNA substrate. After incubation for 16 hours there is no detectable degradation of the DNA substrate as determined by agarose gel electrophoresis.

    Certificate of Analysis

    The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality control's for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]


    The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

    Safety Data Sheet

    The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.