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  • GpC Methyltransferase (M.CviPI)

    Description

    The GC Methyltransferase, M.CviPI, methylates all cytosine residues (C5) within the double-stranded dinucleotide recognition sequence 5´...GC...3´.

    Product Source

    The GpC Methyltransferase, M.CviPI, is isolated from a strain of E. coli which contains the methyltransferase gene from Chlorella virus. This construct is fused to the maltose binding protein (MBP).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    GC Reaction Buffer10X
    S-adenosylmethionine (SAM)-2032 mM

    Advantages and Features

    Applications

    • Blocking restriction endonuclease cleavage
    • Altering the physical properties of DNA
    • Uniform [3H]-labeling of DNA

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme required to protect 1 µg of λ DNA in a total reaction volume of 20 µl in 1 hour at 37°C against cleavage by HaeIII restriction endonuclease. 

    Reaction Conditions

    1X GC Reaction Buffer
    Supplement with 160 μM S-adenosylmethionine (SAM)
    Incubate at 37°C

    M.CviPI is incubated with 1 µg λ DNA in 20 µl 1X GC Reaction Buffer and 160 µM S-adenosylmethionine, for one hour at 37°C. The extent of protection by M.CviPI is determined by the addition of 30 µl NEBuffer 2 containing 10 units of HaeIII restriction endonuclease. Incubation for 1 hour at 37°C is followed by analysis on an agarose gel.

    Storage Temperature

    -20°C

    Storage Conditions

    15 mM Tris-HCl
    0.2 mM NaCl
    1 mM DTT
    0.1 mM EDTA
    200 μg/ml BSA
    50% Glycerol
    pH 7.4 @ 25°C

    Heat Inactivation

    65°C for 20 min

    Quality Control

    Quality Assurance Statement

    • Purified free of contaminating exonucleases and endonucleases.

    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, Buffer):
      The buffer is tested 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

      • S-adenosylmethionine (SAM) is supplied as a 32 mM solution in 0.005 M sulfuric acid and 10% ethanol. Under these conditions SAM is stable for up to 6 months when stored at -20°C.
      • SAM is unstable at (pH 7.5), 37°C, and should be replenished in reactions incubated longer than 4 hours.
    1. Requires fresh DTT for optimum activity. For best results, mix assay buffer fresh for each use.
    2. Methylation at cytosine residues has also been shown to affect the physical properties of DNA, including lowering the free energy of Z-DNA formation (1), increasing the helical pitch of DNA (2), and altering the kinetics of cruciform extrusion (3). Positions of 5-methylcytosine can be identified due to decreased reactivity to hydrazine in chemical sequencing protocols (4).

    References

    1. Zacharias, W. et al. (1988). Biochemistry. 27, 2970-2978.
    2. Gruenbaum, Y. et al. (1982). Nature. 295, 620-621.
    3. Murchie, A.I. and Lilley, D.M. (1989). J. Mol. Biol.. 205, 593-602.
    4. Ohmori, H. et al. (1978). Nucl. Acids. Res. 5, 1479-1485.
    5. Xu, S. et al. (1998). Nucl. Acids Res.. 26, 3961-3966.
    6. Kladde, M.P. et al. (1991). Methods Enzymol. . 304, 431-447.

    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.
    Make sure the reaction buffer is diluted out just before doing the methylation reaction. This enzyme requires fresh DTT for optimal activity.