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  • TspMI


    Product Source

    Thermus species (P. Sharma)

    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 pUCAdeno plasmid DNA in 1 hour at 75°C in a total reaction volume of 50 µl.

    Reaction Conditions

    1X CutSmart® Buffer
    Incubate at 75°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: 75%
    NEBuffer 3.1: 50%
    CutSmart® Buffer: 100%

    Diluent Compatibility

    Storage Temperature


    Storage Conditions

    20 mM Tris-HCl
    300 mM NaCl
    1 mM DTT
    1 mM EDTA
    200 μg/ml BSA
    50% Glycerol
    0.1% Triton® X-100
    pH 8.0 @ 25°C

    Heat Inactivation


    Methylation Sensitivity

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

    Activity at Temperature

    @37°C: 20%
    @65°C: 100%


    1. May exhibit star activity in NEBuffer 1.1, NEBuffer 2.1 or NEBuffer 3.1.
    2. Temperature activity curve of activity:
      37°C gives 20% optimal activity
      65°C gives 100% optimal activity
      75°C gives 100% optimal activity
      80°C gives 200% optimal activity
    3. TspMI is an isoschizomer of XmaI.
    4. For enzymes that cannot be heat-inactivated, we recommend using a column for cleanup (such as the Monarch® PCR & DNA Cleanup Kit), or running the reaction on an agarose gel and then extracting the DNA (we recommend Monarch Gel Extraction Kit), or performing a phenol/chloroform extraction.


    1. How can I search for a restriction enzyme by sequence, overhang or name?
    2. How should I stop my restriction digest?
    3. How stable is a particular restriction enzyme?
    4. When is star activity a concern?
    5. How should I set up a restriction digest?
    6. I don't see any cleavage after my restriction digest. What factors can interfere with cleavage?
    7. How can I generate a restriction enzyme site map for my sequence?
    8. What information is available in the Restriction Enzyme Database (REBASE)?
    9. Do degenerate recognition sites need to be palindromic?
    10. Is extended digestion (incubation times > 1 hour) recommended?
    11. What effect does BSA have on the performance of NEB’s restriction enzymes when included in the new buffers?
    12. Do I have to set-up digests with Time-Saver™ qualified enzymes for 5-15 minutes? Can I digest longer?
    13. How can I access the old NEBuffer Activity Chart?
    14. 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?
    15. What does it mean to be Time-Saver™ qualified?
    16. Why do I see additional DNA bands on my gel after a restriction digest?
    17. Why do I see a DNA smear on an agarose gel after a restriction digest?
    18. Why is my Restriction Enzyme not cutting DNA?
    19. How many nucleotides do I have to add adjacent to the RE recognition site in order to get efficient cutting?

    Tech Tips

    Enzyme is an isoschizomer of Xma I

    Blocked by CpG methylation


    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.
    • 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.