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  • DNA Gyrase (E. coli)

    Description

    DNA Gyrase (E. coli) is a Type II topoisomerase that catalyzes the introduction of negative supercoils in DNA in the presence of ATP. The gyrase holoenzyme is a heterotetramer made up of 2 gyrA (97 kDa) subunits and 2 gyrB (90 kDa) subunits.

    Product Source

    An E. coli strain containing the cloned gyrA and gyrB genes.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    DNA Gyrase (E. coli) Reaction Buffer5X

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme that catalyzes the conversion of DNA Gyrase (E. coli) Substrate NEB #N0471 to >95% of 0.5 μg of supercoiled plasmid in a total reaction volume of 30 μl in 30 minutes at 37°C.

    Reaction Conditions

    1X DNA Gyrase (E. coli) Reaction Buffer
    Incubate at 37°C

    1X DNA Gyrase (E. coli) Reaction Buffer:
    35 mM Tris-HCl
    24 mM KCl
    4 mM MgCl2
    2 mM DTT
    1.75 mM ATP
    5 mM spermidine
    0.1 mg/ml BSA
    6.5% Glycerol
    pH 7.5 @ 25°C

    Storage Temperature

    -70°C

    Storage Conditions

    10 mM Tris-HCl
    50 mM KCl
    2 mM DTT
    50% Glycerol
    0.1 mM EDTA
    pH 7.5 @ 37°C

    Heat Inactivation

    65°C for 20 min

    Supplied As

    Liquid

    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.
    • 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.
    • 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. The 5X DNA Gyrase Reaction Buffer should be stored at -70°C to maintain optimal stability of components.

    References

    1. Adachi, T. et al. (1987). Nucleic Acids Res.. 15, 771-784.
    2. Higgins, N.P. et al. (1978). PNAS. 75, 1773-1777.
    3. Swanberg, S.L. and Wang, J.C. (1987). J. Mol. Biol.. 197, 729-736.

    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. Is the linking number (ΔLk) or the superhelical density (σ) that results from the gyrase reaction fixed or random? Does the sequence, GC content or size of the circular DNA affect the linking number?
    2. Will E. coli DNA Gyrase add additional negative supercoils in already supercoiled DNA?
    3. Do you offer a reverse gyrase?
    4. How should a DNA treated with gyrase be run on a gel?
    5. Can E. coli DNA Gyrase supercoil close and open circular DNA?
    1. Standard E. coli DNA Gyrase Reaction (M0306)

    Usage Guidelines & Tips