mRNA Cap 2'-O-Methyltransferase

mRNA Cap 2´ O Methyltransferase specifi cally requires a Cap 0 structure (m7Gppp5'N) as a substrate, which synthesis from in vitro transcripts mRNA and capping enzyme modify. This enzyme utilizes a methyl donor such as SAM to add a methyl group at the 2’ -O position forming cap-1 structure.
No. Size Price Qty Status
C15038-2000U set $100.00 Inquiry
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Escherichia coli
>95% as determined by SDS-PAGE. Purified by Ni-NTA chromatography.
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 Condition:
1X Capping enzyme reaction buffer, supplemented with 0.5 mM GTP and 0.1 mM S-adenosylmethione (SAM). Incubate at 37°C.
10X Capping enzyme Reaction Buffer: 500 mM Tris-HCl (pH 8.0), 50 mM KCl, 10 mM MgCl2, 10 mM DTT.
Storage Buffer:
Capping enzyme is supplied in 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 0.1% Triton® X-100 and 50% Glycerol.

Stored at -20°C. Avoid repeated freeze/thaw cycles..

Reaction should be performed under RNase free condition. Use nuclease-free tubes, reagents and water to avoid RNase contamination. Also, wear gloves when working with RNA.
One-Step Capping and 2’-O-Methylation procedures:
1. Combine RNA and nuclease-free H2O to a final 14 μL.
2. Heating at 65°C for 5 minutes then chill on ice for 5 minutes.
3. Below reaction mixture should be prepared on ice and combined in the following order:
Component Amount Final concentration
Denatured uncapped RNA 14 μL -
10X Capping Buffer 2 μL 1X
10 mM GTP 1 μL 0.5 mM
4 mM SAM 1 μL 0.2 mM
Vaccinia Capping Enzyme 1 μL 10 U/rxn
mRNA Cap 2´-O-Methyltransferase 1 μL 50 U/rxn
4. Gently mix the reaction thoroughly to achieve uniform distribution.
5. Incubate at 37°C for 60 minutes (For RNA less than 200 nt long increase incubation time to 2 hours).