mRNA Cap 2´-O-Methyltransferase
Cat. No.: SB-G3455-2000U
Size: 2000U
Description
mRNA Cap 2´-O-Methyltransferase is derived from vaccinia virus mRNA Cap 2´-O-methyltransferase, which is recombinantly expressed in Escherichia coli and can add a methyl group to the 2'-O position of the first nucleotide adjacent to the cap structure at the 5' end of the RNA to form a Cap-1 structure. The Cap-1 structure can enhance the translation efficiency of mRNA, so it can help to improve the expression in mRNA transfection and microinjection experiments. mRNA Cap 2´-O-Methyltransferase requires RNA with a m7GpppN cap as a substrate. Mainly used for in vitro mRNA synthesis.
- Source: derived from vaccinia virus, recombinantly expressed by Escherichia coli.
- Definition of enzyme activity: at 37°C, the amount of enzyme required to methylate 10 pmol of 80 nt capped RNA transcript within 1 h is defined as one enzyme activity unit.
- Purity and concentration: SDS-PAGE detection purity > 95%; endogenous nucleic acid residue < 1 pg/μL (qPCR detection); 50 U/μL.
- Enzyme storage buffer: 20 mM Tris-HCl, 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol, 0.1% (w/v) Triton X-100, pH 8.0.
- 10х Capping buffer: 500 mM Tris-HCl, 50 mM KCl, 10 mM MgCl2, 10 mM DTT, pH 8.0.
Storage and Handling Conditions
Transport with wet ice; Store at -20℃, valid for 12 months.
Composition
| Component Number |
Component |
G3455-2000U |
| G3455-1 |
mRNA Cap 2´-O-Methyltransferase |
40 μL |
| G3455-2 |
10х Capping Buffer |
200 μL |
| G3455-3 |
SAM (32 mM) |
20 μL |
| G3455-4 |
Nuclease-Free water |
1 mL |
| Product Manual |
1 copy |
Steps
1. Using Nuclease-Free water, dilute an appropriate amount (within 10 μg) of capped Cap 0 RNA to 16 μL, and heat at 65°C for 5 min;
2. Place the heated centrifuge tube on ice for 5 minutes;
3. Add the components according to the table below:
| Component |
Volume |
| Denatured Cap 0 RNA after processing |
16 μL |
| 10х Capping Buffer |
2 μL |
| SAM (2mM) |
1 μL |
| mRNA Cap 2´-O-Methyltransferase |
1 μL |
4. Incubate the system at 37°C for 1 hour, and the methylation of RNA can be completed; (reaction time can be adjusted)
5. If the capped RNA needs to be poly(A) tailed, Poly(A) RNA Polymerase can be used.
Precautions
- Involving RNA operation, it is necessary to strictly follow the specifications of RNA operation to avoid RNase contamination. Relevant reagents and consumables need to be treated with DEPC to remove RNase or ensure that they are RNase free.
- Reaction heat treatment of RNA is used to remove the secondary structure at the 5' end. If the structure of the 5' end is complex, the time can be extended to 10 min.
- If the capping effect is not good, the reaction time can be extended to 1 h to improve the capping efficiency.
- SAM is unstable at pH 7-8 and 37°C, and needs to be freshly prepared before the reaction. Dilute 32mM SAM to 4 mM working solution before starting the reaction. To avoid SAM degradation, the whole process needs to be placed on ice.
- It is recommended to apply RNase inhibitors to prevent RNA degradation during the reaction. An RNase inhibitor ( G3414 ) can be added at a final concentration of 1 U/μL .
- For your safety and health, please wear a lab coat and disposable gloves for operation.
