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  • MspJI

    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
    R0661S200 units5,000 units/ml$103.00Add to Cart
    R0661L1,000 units5,000 units/ml$412.00Add to Cart
    Epigenetic Analysis (EpiMark® Validated) Products,
    Methylation Dependent Restriction Enzymes for Epigenetics,
    Restriction Endonucleases: H-M
    Methylated DNA Analysis,
    Restriction Enzyme Digestion,
    Restriction Enzymes for Epigenetics


    MspJI, an EpiMark®, validated product is a modification-dependent endonuclease that recognizes mCNNR sites and generates a double-stranded DNA break on the 3´ side of the modified cytosine at N9/N13. The 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 MspJI.

    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 MspJI.

    At fully methylated CpG sites:
    5' . . . Y N mC G N R . . . 3'
    3' . . . R N G mC N Y . . . 5'

    or CHG sites:
    5' . . . Y mC H G R . . . 3'
    3' . . . R G D mC Y . . . 5'

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

    MspJI 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. MspJI does not cleave unmodified DNA.

    Product Source

    An E. coli strain that carries the synthetic MspJI gene from Mycobacterium species JLS.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    NEBuffer 4-2010X
    Enzyme Activator Solution30X

    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 1X 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 NEBuffers2

    NEBuffer 1.1: 10%
    NEBuffer 2.1: 10%
    NEBuffer 3.1: 10%
    CutSmart™ Buffer: 50%

    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. Use of excess enzyme inhibits cleavage. Optimization of the amount of enzyme needed for complete digestion may be required for each substrate DNA. Excess of enzyme or prolonged digestion time in the presence of Enzyme Activator Solution may cause star activity.
    2. Star activity may result from extended digestion


    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. How can I access the old NEBuffer Activity Chart?
    2. How can I access the old Double Digest Finder?
    1. Genomic DNA Digestion using MspJI (R0661)
    2. Optimizing Restriction Endonuclease Reactions
    3. Double Digest Protocol with Standard Restriction Enzymes

    Selection Tools

    Usage Guidelines & Tips

    Troubleshooting Guides

    Interactive Tools

    NEB Publications

    • Xiaojun Huang, Hanlin Lu, Jun-Wen Wang, Liqin Xu, Siyang Liu, Jihua Sun, Jun Wang, Fei Gao (2013) BMC Genetics 14:56, PubMedID: 23773292, DOI: 10.1186/1471-2156-14-56