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  • dam Methyltransferase

    Description

    dam Methyltransferase modifies the adenine residue (N6) of the sequence GATC.

    Product Source

    An E. coli strain that carries plasmid pTP166 carrying the dam modification gene of E. coli

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    dam 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 of λ DNA in 1 hour at 37°C in a total reaction volume of 10 µl against cleavage by MboI restriction endonuclease.

    Reaction Conditions

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

    1X dam Methyltransferase Reaction Buffer:
    50 mM Tris-HCl
    5 mM β-ME
    10 mM EDTA
    pH 7.5 @ 25°C

    dam Methyltransferase is incubated with 1 µg of λ DNA in 10 µl of 1X dam Methyltransferase Buffer, supplemented with 80 µM S-adenosylmethionine, for 1 hour at 37°C followed by 15 minutes at 65°C. The extent of protection is determined by addition of 40 µl 1X NEBuffer 3 supplemented with 10 mM MgCl2 and 10 units of MboI restriction endonuclease. Incubation at 37°C for 1 hour is followed by analysis on an agarose gel.

    Storage Conditions

    50 mM Tris-HCl
    50 mM KCl
    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.
    • Functional Test (Methyltransferase):

      The Methyltransferase is tested in a reaction with Lambda DNA and SAM, followed by a second reaction with the appropriate restriction enzyme. After incubation the extent of methylation is determined by agarose gel electrophoresis.

    • 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.

    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 dam Methyltransferase be heat inactivated?
    4. Does dam Methyltransferase require MgCl2?
    5. Is dam Methyltransferase sensitive to salt?
    6. Will DpnI cleave dam methylated DNA?

    Usage Guidelines & Tips

    Always add the SAM to the reaction buffer just before doing the methylation reaction, because the SAM is unstable in an aqueous solution.

    Keep the SAM frozen at -20C for a longer shelf life, fresh SAM is important for optimal methylation reactions.