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  • T4 Phage β-glucosyltransferase (T4-BGT)

    Description

    T4 Phage β-glucosyltransferase specifically transfers the glucose moiety of uridine diphosphoglucose (UDP-Glc) to the 5-hydroxymethylcytosine (5-hmC) residues in double-stranded DNA, making beta-glucosyl-5-hydroxymethylcytosine (1,2).

    Product Source

    An E. coli strain that carries the cloned bgt gene from bacteriophage T4

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    NEBuffer 4-2010X
    Uridine Diphosphate Glucose50X

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme required to protect 0.5 µg T4gt-DNA against cleavage by MfeI restriction endonuclease.

    Protection Unit Assay Conditions:
    0.5 µg T4gt-DNA, 1X NEBuffer 4 and 40 µM UDP-Glucose in a 30 µl reaction. Incubate for 1 hour at 37°C followed by 10 minutes at 65°C. The extent of protection by T4 -BGT is determined by the addition of 20 µl 1X NEBuffer 4 and 10 units of MfeI. Incubation at 37°C for 30 minutes is followed by analysis on agarose gels.

    Enzyme Properties
    Activity in NEBuffers:
    NEBuffer 1 100
    NEBuffer 2 50
    NEBuffer 3 50
    NEBuffer 4 100% 

    Survival in a Reaction: A minimum of 0.16 unit for 16 hours is required to protect 0.5 µg T4 gt-DNA against cleavage by MfeI.

    Reaction Conditions

    1X NEBuffer 4
    Incubate at 37°C

    1X NEBuffer 4:
    50 mM Potassium Acetate
    20 mM Tris-acetate
    10 mM Magnesium Acetate
    1 mM DTT
    pH 7.9 @ 25°C

    Storage Temperature

    -20°C

    Storage Conditions

    20 mM KPO4
    200 mM NaCl
    0.25 mM DTT
    0.1 mM EDTA
    50% Glycerol
    pH 7.0 @ 25°C

    Heat Inactivation

    65°C for 10 min

    Molecular Weight

    Theoretical: 40666 kDa

    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.

    References

    1. Josse, J. and Kornberg, A. (1962). J. Biol.Chem.. 237, 1968-1976.
    2. Tomaschewski, J. et al. (1985). Nucleic Acids Res. 13, 7551-7568.
    3. McNicol, L. A. et al. (1973). J. Mol. Biol. 15, 76,, 285-301.
    4. Szwagierczak, A. et al. (2010). Sensitive enzymatic quantification of 5-hydroxymethylcytosine in genomic DNA. Nucleic Acids Res. 38, e181.

    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.

    Citations

    • Bhattacharyya, S., Yu, Y., Suzuki, M., Campbell, N., Mazdo, J., Vasanthakumar, A., et al. (2013) Genome-wide hydroxymethylation tested using the HELP-GT assay shows redistribution in cancer Nucleic Acids Research DOI: doi:10.1093/nar/gkt601