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  • FokI

    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 incubation temp heat inactivation cpg dcm
    Fok-I-cutsite_1
    Catalog #SizeConcentrationPriceQtyAdd to Cart
    R0109S1,000 units5,000 units/ml$63.00Add to Cart
    R0109L5,000 units5,000 units/ml$252.00Add to Cart
      
    Categories:
    Restriction Endonucleases: C-G
    Applications:
    Restriction Enzyme Digestion

    Description

    FokI crystals (Ira Schildkraut and Lydia Dorner, New England Biolabs)

    Product Source

    An E. coli strain that carries the FokI gene from Flavobacterium okeanokoites (IFO 12536).

    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: 100%
    NEBuffer 2.1: 100%
    NEBuffer 3.1: 75%
    CutSmart™ Buffer: 100%

    Diluent Compatibility

    Storage Temperature

    -20°C

    Storage Conditions

    10 mM Tris-HCl
    50 mM NaCl
    1 mM DTT
    0.1 mM EDTA
    200 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: Impaired by Overlapping
    CpG Methylation: Impaired by Overlapping

    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.
    • 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.

    Notes

    1. FokI can cleave between virtually any two nucleotides by constructing a complementary oligonucleotide to the sequence to be cleaved (Szybalski, W. (1985) Gene 40, 169-173, Podhajska, A. and Szybalski, W. (1985) Gene 40, 175-182).
    2. Overdigestions of >5 units of FokI per μg of DNA and incubation times >2 hours are not recommended.
    3. Star activity may result from a glycerol concentration of >5%

    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]

    Specifications

    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.

    Datacards

    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. Does FokI tend to degrade DNA?
    2. Why does FokI degrade DNA?
    3. Is extended digestion of FokI recommended?
    4. Does FokI have trouble cleaving PCR products?
    5. Is FokI blocked by methylation?
    6. Is FokI used in special techniques?
    7. What is the molecular weight of FokI?
    8. Is FokI activity sensitive to pH?
    9. Is FokI active at 25°C?
    10. Does FokI cleave ssDNA?
    11. My enzyme is no longer Time-Saver™ qualified. What happened?
    12. What effect does BSA have on the performance of NEB’s restriction enzymes when included in the new buffers?
    13. How can I access the old NEBuffer Activity Chart?
    14. How can I access the old Double Digest Finder?
    1. Optimizing Restriction Endonuclease Reactions

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