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  • T7 Endonuclease I

    Description

    T7 Endonuclease I recognizes and cleaves non-perfectly matched DNA, cruciform DNA structures, Holliday structures or junctions, heteroduplex DNA and more slowly, nicked double-stranded DNA. The cleavage site is at the first, second or third phosphodiester bond that is 5´ to the mismatch. The protein is the product of T7 gene 3.

    Highlights

    • Isolated from a recombinant source
    • Supplied with 10X Reaction Buffer
    • Recognizes non-perfectly matched DNA

    Product Source

    An E. coli strain that carries a fusion of maltose binding protein and T7 Endonuclease I (tT7 Endo I).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    NEBuffer 2-2010X

    Advantages and Features

    Applications

    • Resolve four-way junction or branched DNA
    • Detect or cleave heteroduplex and nicked DNA
    • Randomly cleave linear DNA for shot-gun cloning

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme required to convert > 90% of 1 μg of supercoiled cruciform pUC(AT) to > 90% linear form in a total reaction volume of 50 μl in 1 hour at 37°C.

    Reaction Conditions

    1X NEBuffer 2
    Incubate at 37°C

    1X NEBuffer 2:
    50 mM NaCl
    10 mM Tris-HCl
    10 mM MgCl2
    1 mM DTT
    pH 7.9 @ 25°C

    Storage Temperature

    -20°C

    Storage Conditions

    20 mM Tris-HCl
    200 mM NaCl
    1 mM DTT
    0.1 mM EDTA
    50% Glycerol
    0.15% Triton® X-100
    pH 7.5 @ 25°C

    Heat Inactivation

    No

    Notes

    1. T7 Endonuclease I is a structure-selective enzyme. It acts on a variety of DNA substrates with different specific activities. It is important to control the amount of enzyme and the reaction time used for cleavage of a particular substrate.
    2. Temperatures above 42°C cause an increase in nonspecific nuclease activity and should be avoided.
    3. pUC(AT) is derived from pUC19 with a modification of the polylinker between the EcoRI site and the PstI site.

    References

    1. Xu, M. -Q. and Evans, T.C. (2001). Methods. 24, 257-277.
    2. Parkinson, M.J. and Lilley. D.M.J. (1997). J. Mol. Biol.. 270, 169-178.
    3. White, M.F. et al. (1997). J. Mol. Biol.. 269, 647-664.
    4. Hadden, J.M. et al. (2001). Nat. Struct. Biol.. 8, 62-67.

    FAQs

    1. Does T7 Endonuclease I recognize single base pair mismatches?
    2. What is the activity of T7 Endonuclease I in other buffers?
    3. What is the activity of T7 Endonuclease I on various DNA substrates?
    4. Does Mn enhance T7 Endonuclease I activity?
    5. Can T7 Endonuclease I be heat inactivated?
    6. What is the activity of T7 Endonuclease I at different reaction temperatures?
    7. What common additives inhibit T7 Endonuclease I?
    8. How stable is T7 Endonuclease I in reaction?
    9. What is the molecular weight of T7 Endonuclease I?
    10. Can I use T7 Endonuclease I genome editing (CRIPSR/Cas9,TALEN,ZFN) mismatch detection assays using unpurified PCR products?
    11. What PCR reagents do you recommend for DNA amplification in genome editing (CRIPSR/Cas9,TALEN,ZFN) mismatch detection assays?

    Protocols

    1. Determining Genome Targeting Efficiency using T7 Endonuclease I

    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.

    Selection Tools

    Usage Guidelines & Tips

    Citations

    • A-Bk Dad, S Ramakrishna, M Song, H Kim (2014). Enhanced gene disruption by programmable nucleases delivered by a minicircle vector. Gene Ther. , PubMedID: 25142139, DOI: 10.1038/gt.2014.76

    Quality Control

    Quality Assurance Statement

    • Purified free of contaminating exonucleases and endonucleases.

    Safety Data Sheet

    The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

    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.