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  • FspEI

    This enzyme has transitioned to an improved new buffer system. Visit NEBCutSmart.com for further details.
    The new and current Double Digest Finder and current Activity/Performance Chart for the CutSmart buffer system are available. The previous version of the Double Digest Finder, as well as the previous Version of Activity/Performance Chart that use the former buffer system, are still available for your convenience.
    cloned at neb recombinant NEBuffer 4 incubation temp heat inactivation bsa
    Catalog #SizeConcentrationPriceQtyAdd to Cart
    R0662S200 units5,000 units/ml$103.00Add to Cart
    R0662L1,000 units5,000 units/ml$412.00Add to Cart
    Methylation Dependent Restriction Enzymes,
    Restriction Endonucleases: C-G,
    Restriction Enzymes for Epigenetic Analysis (EpiMark® Validated)
    Methylated DNA Analysis,
    Restriction Enzyme Digestion,
    Restriction Enzymes for Epigenetics


    FspEI, an EpiMark® validated product, is a modification-dependent endonuclease which recognizes CmC sites and generates a double-stranded DNA break on the 3´ side of the modified cytosine at N12/N16. Recognized cytosine modifications include C5-methylation (5-mC) and C5-hydroxymethylation (5-hmC) (1). 

    This enzyme is provided with an Enzyme Activator Solution which may be used for efficient digestion by FspEI.

    The most common epigenetic modifications found in eukaryotic organisms are methylation marks at CpG or CHG sites. A subset of these modified sites are recognized and cleaved by FspEI. 

    At fully methylated CpG sites: 
    5´. . . C mC  G G . . . 3´
    3´. . . G  G mC C . . . 5´

    or CHG sites: 
    5´. . . C mC H  G G . . . 3´
    3´. . . G  G D mC C . . . 5´

    H = A or C or T (not G)
    D = A or G or T (not C) 

    FspEI recognizes each hemi-methylated site individually and cleaves bidirectionally to generate 32 base or 31 base fragments, respectively. These fragments contain the central methylated site and have 4-base 5´ overhangs at each end. FspEI does not cleave unmodified DNA.

    Product Source

    An E. coli strain that carries the synthetic FspEI gene from Frankia species EAN1pec.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    NEBuffer 4-2010X
    Enzyme Activator Solution30X
    BSA-2010 mg/ml

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme required to digest 1 µg of pBR322 (dcm+) DNA in 1 hour at 37°C in a total reaction volume of 50 µl.

    Reaction Conditions

    1X NEBuffer 4
    Supplement with BSA and 1X Enzyme Activator Solution
    Incubate at 37°C

    1X NEBuffer 4:
    50 mM Potassium Acetate
    20 mM Tris-acetate
    10 mM Magnesium Acetate
    1 mM DTT
    pH 7.9 @ 25°C

    Activity in NEBuffers

    NEBuffer 1.1: 10%
    NEBuffer 2.1: 10%
    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: Not Sensitive
    dcm methylation: Not Sensitive
    CpG Methylation: Not Sensitive

    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.
    • 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.
    • 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.
    • RNase Activity (1 Hour Digestion):
      The product is tested in a reaction containing a RNA substrate.  After incubation for 1 hour there is no detectable degradation of the RNA substrate as determined by gel electrophoresis.


    1. Zheng, Y. et al. (2010). Nucl. Acids Res. doi:10, 1093/nar/gkq327.
    2. U.S. Publication No. 2010-0167942 Unpublished observation

    Supporting Documents

    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]

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.


    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. What effect does BSA have on the performance of NEB’s restriction enzymes when included in the new buffers?
    2. How can I access the old NEBuffer Activity Chart?
    3. How can I access the old Double Digest Finder?
    1. Genomic DNA Digestion using FspEI (R0662)
    2. Optimizing Restriction Endonuclease Reactions