• My NEB
  • Print
  • PDF
  • NotI

    Did you know this product can be customized or purchased in larger volumes? Submit an inquiry to find out more about customization options.
    cloned at neb recombinant timesaver 5min incubation temp heat inactivation cpg
    Catalog #SizeConcentrationPriceQtyAdd to Cart
    R0189S500 units10,000 units/ml$70.00Add to Cart
    R0189L2,500 units10,000 units/ml$282.00Add to Cart
    R0189M2,500 units50,000 units/ml$282.00Add to Cart
    Restriction Endonucleases: N-O,
    Time-Saver™ Qualified Restriction Enzymes
    Restriction Enzyme Digestion


    NotI has a High Fidelity version NotI-HF® (NEB #R3189).

    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 minutes 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.

    For details on NEB’s quality controls for restriction endonucleases, visit our Restriction Enzyme Qualit y page.

    Product Source

    An E. coli strain that carries the cloned NotI gene from Nocardia otitidis-caviarum (ATCC 14630).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    NEBuffer 3.1-2010X

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme required to digest 1 µg of pBC4 DNA in 1 hour at 37°C in a total reaction volume of 50 µl.

    Reaction Conditions

    1X NEBuffer 3.1
    Incubate at 37°C

    1X NEBuffer 3.1:
    100 mM NaCl
    50 mM Tris-HCl
    10 mM MgCl2
    100 μg/ml BSA
    pH 7.9 @ 25°C

    Activity in NEBuffers

    NEBuffer 1.1: 10%
    NEBuffer 2.1: 50%
    NEBuffer 3.1: 100%
    CutSmart® Buffer: 25%

    Diluent Compatibility

    Storage Temperature


    Storage Conditions

    10 mM Tris-HCl
    250 mM NaCl
    1 mM DTT
    0.1 mM EDTA
    200 μg/ml BSA
    50% Glycerol
    0.15% Triton® X-100
    pH 7.4 @ 25°C

    Heat Inactivation

    65°C for 20 min

    Methylation Sensitivity

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


    1. Samuelson, J.C., Morgan, R.D., Benner, J.S., Claus, T.E., Packard, S.L. and Xu, S.Y. (2006). Engineering a rare-cutting restriction enzyme: Genetic screening and selection of NotI variants. Nucleic Acids Research. 34, 796-805. PubMedID: 16456032
    2. Zhang, P., Bao, Y., Higgins, L. and Xu, S.Y. (2007). Rational design of a chimeric endonuclease targeted to NotI recognition site. Protein Engineering, Design and Selection. 20, 497-504.
    3. Lambert, A.R., Sussman, D., Shen, B., Maunus, R., Nix, J., Samuelson, J., Xu, S.Y. and Stoddard, B.L. (2008). Structures of the Rare-Cutting Restriction Endonuclease NotI Reveal a Unique Metal Binding Fold Involved in DNA Binding. Structure. 16, 558-569.


    1. What effect does BSA have on the performance of NEB’s restriction enzymes when included in the new buffers?
    2. Do I have to set-up digests with Time-Saver™ qualified enzymes for 5-15 minutes? Can I digest longer?
    3. How can I access the old NEBuffer Activity Chart?
    4. Why is my Restriction Enzyme not cutting DNA?
    5. Why do I see a DNA smear on an agarose gel after a restriction digest?
    6. Why do I see additional DNA bands on my gel after a restriction digest?
    7. 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.
    • 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.