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  • Topoisomerase I (E. coli)

    Description

    Topoisomerase I (E. coli) catalyzes the relaxation of negatively supercoiled DNA. Topoisomerase I has also been implicated in knotting and unknotting DNA (1) and in linking complementary rings of single-stranded DNA into double-stranded rings (2). The intact holoenzyme is a 97 kDa protein.

    Highlights

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

    Product Source

    An E. coli strain containing the cloned topA gene

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    BSA-2010 mg/ml
    CutSmart™ Buffer-2010X

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme that catalyzes the relaxation of  > 95% of 0.5 μg of pUC19 RF I (negatively supercoiled) DNA in 15 minutes at 37°C in a total reaction volume of 25 μl. DNA supercoiling is assessed by agarose gel electrophoresis in the absence of ethidium bromide.

    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

    Storage Temperature

    -20°C

    Storage Conditions

    10 mM Tris-HCl
    35 mM (NH4)2SO4
    50 mM KCl
    1 mM DTT
    0.1 mM EDTA
    50% Glycerol
    pH 7.5 @ 25°C

    Heat Inactivation

    65°C for 20 min

    References

    1. Liu, L.F., Depew, R.E. and Wang, J.C. (1976). J. Mol. Biol.. 106, 439-452.
    2. Kirkegaard, K. and Wang, J.C. (1978). Nucl. Acids Res.. 5, 3811-3820.

    Supporting Documents

    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 Topoisomerase I relax all supercoiled DNA to the same degree or are the products an equilibrium of various conformations?
    2. If the DNA resulting from a Topoisomerase I reaction is run in an ethidium bromide gel and there seems to be no evidence of enzyme activity, what is the most probable explanation?
    3. What is the molecular weight of Topoisomerase I?
    4. Can Topoisomerase I relax both positively and negatively supercoiled DNA?
    5. Does E. coli Toposiomerase I have the same properties as the Vaccinia Topoisomerase I?
    6. Will Topoisomerase I produce single strand-breaks in a plasmid?

    Usage Guidelines & Tips