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Product description: mRNA Cap-2'-O-Methyltransferase is a recombinant protein from Vaccinia virus. The enzyme can add 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 SAM as a methyl donor to methylate capped RNA(Cap 0) resulting in a cap-1 structure. The Cap-1structure has been reported to enhance mRNA translation efficiency 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.
Source: Recombinant E.coli
Concentration and Size: 50U/μL
Unit Definition: One unit is defined as the amount of enzyme required to methylate 10pmols of 80 nt Capped RNA transcript in 1 hour at 37℃.
Storage Buffer: 20mM Tris-HCl (pH8.0), 0.1mM EDTA, 100mM NaCl, 1mM DTT, 0.1%(v/v) Triton X-100, 50%(v/v) glycerol.
Companion Product: 10X Capping Buffer, Cat#ON-073, 500mM Tris-HCl, pH8.0, 50mM KCl, 10mM MgCl2, 10mM DTT; S-adenosylmethionine (SAM),32mM, Cat#ON-074
1. Do not resuspend the RNA in an EDTA-containing solution.
2. To enhance the stability of RNA in solution, RNase inhibitor is desired, which can be add to a final concertation of 1U/ul at the time of reaction set-up.
3. SAM is unstable at pH 7-8, 37℃ and should be mixed fresh for each reaction series. We recommend determining how many reactions will be performed and diluting an aliquot of the 32mM stock to 2mM.
4. Heating the solution of RNA prior to incubation with Vaccinia capping enzyme, to remove secondary structure on the 5' end of the transcripts.?For highly structured 5' ends, increase the RNA heat-denaturation conditions used. For example, 65℃ for 20 min, 75℃ for 10 min, 85℃ for 5 min, etc…
5. Vaccinia Capping Enzyme and 2'-O-methyltransferase work together to produce the Cap 1 structure.
6. Capping reaction incubation time can be Increased up to 3 hours at 37℃ for a high capping efficiency for transcripts with known structured 5' ends.
Storage Conditions: -20℃
Quality Assurance: Free of Endonuclease, Exonuclease, and RNase activities.
Physical Purity: ＞95% by SDS-PAGE.
1. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 250, No. 24, Issue of December 25, pp. 9322-9329,1975.
2. Kuge, H. et al., (1998) Nucl. Acids Res. 26, 3208.