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  • EcoRI Methyltransferase

    Description

    EcoRI Methyltransferase modifies the internal adenine residue (N6) of the sequence GAATTC.

    Product Source

    An E. coli. strain that carries the cloned EcoRI modification gene from Escherichia coli RY13 (R. N. Yoshimori).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    EcoRI Methyltransferase Reaction Buffer-2010X
    S-adenosylmethionine (SAM)-2032 mM

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme required to protect 1 μg λ DNA in 1 hour at 37°C in a total reaction volume of 10 μl against cleavage by EcoRI restriction endonuclease

    Reaction Conditions

    1X EcoRI Methyltransferase Reaction Buffer
    Supplement with 80 μM S-adenosylmethionine (SAM)
    Incubate at 37°C

    1X EcoRI Methyltransferase Reaction Buffer:
    50 mM Tris-HCl
    50 mM NaCl
    10 mM EDTA
    pH 8 @ 25°C

    EcoRI Methyltransferase is incubated with 1 µg of λ DNA in 10 µl 1X EcoRI Methyltransferase Buffer, supplemented with 80 µM S-adenosylmethionine, for one hour at 37°C followed by 15 minutes at 65°C. The extent of protection by EcoRI Methyltransferase is determined by the addition of 40 µl NEBuffer 2 supplemented with 10 mM MgCl2 and 5 units of EcoRI restriction endonuclease. Incubation at 37°C for 30 minutes is followed by analysis on an agarose gel.

    Storage Temperature

    -20°C

    Storage Conditions

    100 mM KPO4
    200 mM NaCl
    10 mM β-ME
    0.1 mM EDTA
    200 μg/ml BSA
    50% Glycerol
    pH 7.4 @ 25°C

    Heat Inactivation

    65°C for 20 min

    Molecular Weight

    Theoretical: 37911 daltons

    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.
    • 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. Hoffman, J.L. (1986). biochemistry. 25, 4444-4449.

    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 S-adenosylmethionine (SAM) supplied with the Methyltransferase?
    2. What should be considered if the methylation is not going to completion?
    3. Can EcoRI Methyltransferase be heat inactivated?
    4. Does EcoRI Methyltransferase require MgCl2?
    5. Will EcoRI methylation block the ApoI restriction enzyme?

    Usage Guidelines & Tips