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  • BamHI Methyltransferase

    Description

    BamHI Methyltransferase modifies the internal cytosine residue (N4) of the sequence GGATCC.

    Product Source

    An E. coli strain that carries the cloned BamHI modification gene from Bacillus amyloliquefaciens H (ATCC 49763).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    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 BamHI restriction endonuclease.

    Reaction Conditions

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

    BamHI Methyltransferase is incubated with 1 µg λ DNA in 10 µl 1X BamHI 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 BamHI Methyltransferase is determined by the addition of 40 µl NEBuffer 1 supplemented with 10 mM MgCl2 and 10 units of BamHI restriction endonuclease. Incubation at 37°C for 30 minutes is followed by analysis on agarose gels.

    Storage Temperature

    -20°C

    Storage Conditions

    50 mM Tris-HCl
    1 mM DTT
    10 mM EDTA
    200 μg/ml BSA
    50% Glycerol
    pH 7.5 @ 25°C

    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.

    Notes

    1. Storage of SAM: S-adenosylmethionine is stored at –20°C as 32 mM solution dissolved in sulfuric acid (0.005 M) and 10% ethanol. SAM in this solution stored under ideal conditions remains active for up to 6 months. SAM is unstable at (pH 7.5), 37°C, and should be replenished for reactions incubated longer than 4 hours. Many problems in achieving complete digestion can be alleviated by using fresh SAM.

    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. What is the molecular weight of BamHI Methyltransferase?