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  • mRNA Cap 2´-O-Methyltransferase

    Description

    mRNA Cap 2´-O-Methyltransferase adds a methyl group at the 2´-O position of the first nucleotide adjacent to the cap structure at the 5´ end of the RNA. The enzyme utilizes S-adenosylmethionine (SAM) as a methyl donor to methylate capped RNA (cap-0) resulting in a cap-1 structure.

    The cap-1 structure has been reported to enhance mRNA translation efficiency (1) and hence may help improve expression in mRNA transfection and microinjection experiments.

    mRNA Cap 2´-O-Methyltransferase specifically requires RNA with an m7GpppN cap as substrate. It cannot utilize RNA with pN, ppN, pppN or GpppN at the 5´ end (2,3). Capped RNA may be prepared via in vitro transcription using cap analog or through enzymatic capping using the Vaccinia Capping Enzyme (NEB #M2080 ).

    The reagents provided in this pack can be used to methylate up to 400 μg of capped RNA.


    M0366

    Product Source

    An E. coli strain that carries the gene for the Vaccinia mRNA Cap 2´-O-Methyltransferase.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    S-adenosylmethionine (SAM)-2032 mM
    Capping Buffer10X

    Advantages and Features

    Applications

    • 2´-O-methylation of capped mRNA for improved expression during microinjection and transfection experiments.

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme required to methylate 10 pmoles of 80 nt long capped RNA transcript in 1 hour at 37°C.

    Reaction Conditions

    1X Capping Buffer
    Incubate at 37°C

    1X Capping Buffer:
    50 mM Tris-HCl
    5 mM KCl
    1 mM MgCl2
    1 mM DTT
    pH 8 @ 25°C

    Storage Temperature

    -20°C

    Storage Conditions

    20 mM Tris-HCl
    100 mM NaCl
    1 mM DTT
    0.1 mM EDTA
    50% Glycerol
    0.1% Triton® X-100
    pH 8.0 @ 25°C

    Notes

    1. (READ PRIOR TO SETTING UP REACTION)

      RNA prepared using in vitro transcription and cap analog should be purified prior to use and resuspended in nuclease-free water. EDTA and salts should not be present in the solution.

      mRNA Cap 2´-O-Methyltransferase may be directly added to a Vaccinia Capping System (NEB #M2080) reaction. RNA purification is not required in this case.

    2. Heating the RNA at 65°C for 5 minutes prior to incubation with the enzyme removes secondary structure on the 5´ end of the transcript. Extend time to 10 minutes for transcripts with known highly structured 5´ ends.

    3. SAM is unstable at pH 7–8, 37°C and should be mixed fresh prior to starting the reaction. We recommend determining how many reactions will be performed and diluting an aliquot of the 32 mM stock to 4 mM immediately before setting up the reactions. This "working stock" should be kept on ice to prevent degradation of SAM.

    References

    1. Kuge, H. et al. (1998). Nucleic Acids Res. 26, 3208-3214.
    2. Barbosa, E. and Moss, B. (1978). J. Biol. Chem. 253, 7698-7702.
    3. Lockless, S.W. et al. (1998).  Biochemistry. 37, 8564-8574.

    FAQs

    1. What kind of RNA can be methylated using the mRNA Cap 2'-O-Methyltransferase?
    2. I want to use the mRNA Cap 2'-O-Methyltransferase with the Vaccinia Capping System (NEB# M2080) in a one-step reaction to produce cap 1 RNA. Is that possible?
    3. What precautions should be taken while using the mRNA Cap 2'-O-Methyltransferase?

    Protocols

    1. 2´-O-Methylation of Capped RNA (M0366)
    2. One-Step Capping and 2´-O-Methylation (M0366)

    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.

    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.
    • Protein Purity (SDS-PAGE):
      The physical purity is assessed by comparing contaminating protein bands in a concentrated sample to the protein of interest band in a sample of known dilution. The purity is determined by SDS-PAGE.
    • RNase Activity (1 Hour Digestion):
      The product is tested in a reaction containing a RNA substrate.  After incubation for 1 hour there is no detectable degradation of the RNA substrate as determined by gel electrophoresis.

    Specifications

    The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

    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.