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  • Haelll Methyltransferase

    Description

    HaeIII Methyltransferase modifies the internal cytosine residue (C5) of the sequence GGCC.

    Product Source

    An E. coli strain that carries the cloned HaeIII modification gene from Haemophilus aegyptius (ATCC 11116).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    HaeIII Methyltransferase Reaction Buffer10X
    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 HaeIII restriction endonuclease.

    Reaction Conditions

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

    1X HaeIII Methyltransferase Reaction Buffer:
    50 mM Tris-HCl
    50 mM NaCl
    10 mM DTT
    pH 8.5 @ 25°C

    HaeIII Methyltransferase is incubated with 1 µg of λ DNA in 10 µl of 1X HaeIII Methyltransferase Reaction Buffer, supplemented with 80 µM S-adenosylmethionine, for one hour at 37°C followed by 15 minutes at 65°C. The extent of protection is determined by the addition of 40 µl NEBuffer 2 and 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

    50 mM Tris-HCl
    1 mM DTT
    10 mM EDTA
    200 μg/ml BSA
    50% Glycerol
    50 mM KCl
    pH 7.4 @ 25°C

    Heat Inactivation

    65°C for 20 min

    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. HaeIII Methyltransferase protects DNA against cleavage by NotI.

    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. Does HaeIII Methyltransferase require DTT?
    4. Can HaeIII Methyltransferase be heat inactivated?
    5. Does HaeIII Methyltransferase require MgCl2?
    6. What is the molecular weight of HaeIII Methyltransferase?