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  • HphI


    Product Source

    An E. coli strain that carries the HphI gene from Haemophilus parahaemolyticus (ATCC 49700).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    CutSmart® Buffer-2010X

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme required to digest 1 µg of λ 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: 50%
    NEBuffer 2.1: 50%
    NEBuffer 3.1: 10%
    CutSmart® Buffer: 100%

    Diluent Compatibility

    Storage Temperature


    Storage Conditions

    10 mM Tris-HCl
    300 mM NaCl
    1 mM DTT
    0.1 mM EDTA
    500 μg/ml BSA
    50% Glycerol
    pH 7.4 @ 25°C

    Heat Inactivation

    65°C for 20 min

    Methylation Sensitivity

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


    1. It has been suggested that HphI may cleave at N9/N8 depending on the sequence between the recognition and cleavage sites (Kang, C. and Wu, C.-W. (1987) Nucl. Acids Res. 15, 2279-2294; Cho, S.-H. and Kang, C. (1990) Mol. Cells 1, 81?86.). Incubation of > 12 units for over 4 hours on ΦX174 DNA results in additional cleavage products. This has not yet been shown to occur on other DNAs. Low pH and high glycerol concentration enhance this activity.
    2. This enzyme is blocked by dam methylation. More information can be found at Dam-Dcm and CpG Methylation.
    3. This enzyme is blocked by dcm methylation. More information can be found at Dam-Dcm and CpG Methylation.


    1. Is there variability in the cleavage site of HphI?
    2. Does HphI exhibit star activity?
    3. What is the activity of HphI  at 25°C?
    4. What effect does BSA have on the performance of NEB’s restriction enzymes when included in the new buffers?
    5. Do I have to set-up digests with Time-Saver™ qualified enzymes for 5-15 minutes? Can I digest longer?
    6. Has the conversion to the new buffer system altered any of the properties of the Restriction Enzyme itself?
    7. How can I access the old NEBuffer Activity Chart?
    8. I tested your restriction enzyme on the substrate DNA recommended by NEB, and it appears to be active, however it does not digest my DNA. What could be the reason?
    9. Why is my Restriction Enzyme not cutting DNA?
    10. Why do I see a DNA smear on an agarose gel after a restriction digest?
    11. Why do I see additional DNA bands on my gel after a restriction digest?
    12. How many nucleotides do I have to add adjacent to the RE recognition site in order to get efficient cutting?

    Tech Tips

    This enzyme will not cut plasmids isolated from most E.coli because it is blocked by dam methylation and dcm methylation. For enzyme cleavage to occur, plasmids must be transformed and isolated from a dam-, dcm- strain of E.coli, such as NEB’s dam-/dcm- Competent E.coli (NEB #C2925).


    1. Optimizing Restriction Endonuclease Reactions
    2. Double Digest Protocol with Standard Restriction Enzymes
    3. Time-Saver Protocol for Restriction Enzyme Digests
    4. Protocol for Direct Digestion of gDNA during droplet digital PCR (ddPCR)
    5. Protocol for Digestion Prior to droplet digital PCR (ddPCR)

    Selection Charts

    Usage Guidelines & Tips

    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.
    • 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.