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  • Hhal Methyltransferase

    Description

    HhaI Methyltransferase modifies the internal cytosine residue (C5) of the sequence GCGC.


    M.HhaI crystal (Xiaodong Cheng, Cold Spring Harbor Laboratory)

    Product Source

    An E. coli strain that carries the cloned HhaI modification gene from Haemophilus haemolyticus (ATCC 10014).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    HhaI Methylase 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 HhaI restriction endonuclease.

    Reaction Conditions

    1X HhaI Methylase Reaction Buffer
    Supplement with 32 mM S-adenosylmethionine (SAM)
    Incubate at 37°C

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

    HhaI Methyltransferase is incubated with 1 µg of λ DNA in 10 µl 1X HhaI 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 HhaI Methyltransferase is determined by the addition of 40 µl NEBuffer 4 supplemented with 100 µg/ml BSA, 10 mM MgCl2 and 10 units of HhaI restriction endonuclease. Incubation at 37°C for 30 minutes is followed by analysis on an agarose gel.

    Storage Temperature

    -20°C

    Storage Conditions

    50 mM Tris-HCl
    150 mM NaCl
    5 mM β-ME
    10 mM EDTA
    200 μg/ml BSA
    50% Glycerol
    pH 7.5 @ 25°C

    Molecular Weight

    Theoretical: 36994 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, DNA):
      The DNA is tested in a reaction under standard reaction conditions. After incubation for 16 hours there is no detectable degradation of the DNA as determined by agarose gel electrophoresis.

    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 HhaI Methyltransferase?