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  • USER™ Enzyme

    Description

    USER (Uracil-Specific Excision Reagent) Enzyme (1) generates a single nucleotide gap at the location of a uracil. USER Enzyme is a mixture of Uracil DNA glycosylase (UDG) and the DNA glycosylase-lyase Endonuclease VIII. UDG catalyses the excision of a uracil base, forming an abasic (apyrimidinic) site while leaving the phosphodiester backbone intact (2,3). The lyase activity of Endonuclease VIII breaks the phosphodiester backbone at the 3´ and 5´ sides of the abasic site so that base-free deoxyribose is released (4,5).

    Product Source

    The two component proteins are purified separately from E. coli K-12 strains containing plasmids encoding Endonuclease VIII and Uracil-DNA Glycosylase.

    Supplied in: 50 mM KCl, 5 mM NaCl, 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 1 mM DTT, 175 mg/ml BSA and 50% glycerol.

    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 nick 10 pmol of a 34 mer oligonucleotide duplex containing a single uracil base, in 15 minutes at 37°C in a total reaction volume of 10 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

    Storage Temperature

    -20°C

    Storage Conditions

    10 mM Tris-HCl
    5 mM NaCl
    1 mM DTT
    0.1 mM EDTA
    175 mg/ml BSA
    50% Glycerol
    50 mM KCl
    pH 7.4 @ 25°C

    Heat Inactivation

    No

    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.
    • Functional Test (USER, Transformation Assay):
      A 10 μl reaction in ThermoPol Reaction Buffer containing 20 ng linearized pNEB206A, 100 ng of a 950 bp control PCR product and 1 unit of USER Enzyme was incubated for 15 minutes at 37°C followed by 15 minutes at 25°C. After transformation into ER2267 chemically-competent cells > 95% of colonies contained recombinant plasmid.

    Notes

    1. USER Enzyme is active in all commercial PCR buffers tested. It also has 100% activity in TE (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA). For further information, see the USER Friendly Cloning Kit manual (NEB #E5500).
    2. Incubate at 37°C.

    References

    1. Bitinaite, J. unpublished observations.
    2. Lindhal, T., Ljungquist, S., Siegert, W., Nyberg, B. and Sperens, B. (1977). J. Biol. Chem.. 252, 3286-3294.
    3. Lindhal, T. (1982). Annu. Rev. Biochem.. 51, 61-64.
    4. Melamede, R.J., Hatahet, Z., Kow, Y.W., Ide, H. and Wallace, S.S. (1994). Biochemistry. 33, 1255-1264.
    5. Jiang, D., Hatahet, Z., Melamede, R.J., Kow, Y.W. and Wallace, S.S. (1997). J. Biol. Chem.. 272, 32230-32239.

    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. Cloning with USER Enzyme

    Usage Guidelines & Tips