mRNA Cap2'-O-Methyltransferase--HYJ232
mRNA Cap 2´-O-Methyltransferase (mRNA Cap 2´-O-Methyltransferase) is derived from a recombinant Escherichia coli strain carrying the vaccinia virus mRNA Cap 2´-O-methyltransferase gene. This enzyme adds a methyl group to the 2´-O position of the first nucleotide immediately preceding the cap structure at the 5´ end of RNA. It can also methylate capped RNA (Cap 0) using S-adenosylmethionine (SAM) as a methyl donor, thereby forming a Cap 1 structure. The Cap 1 structure enhances mRNA translation efficiency and can therefore help improve mRNA expression in transfection and microinjection experiments. This enzyme requires RNA with a 7-methylguanosine cap structure (m7GpppN) as a substrate and cannot be used with RNA containing pN, ppN, pppN, or GpppN at the 5' end. Capped RNA can be prepared by in vitro transcription using a capping analog or enzymatically capped using Vaccinia Capping Enzyme.
Components
|
Components |
2.5KU |
10KU |
50KU |
500KU |
5000KU |
|
mRNA Cap2'-O-Methyltransferase (50 U/μL) |
0.05 mL |
0.2 mL |
1 mL |
10 mL |
100 mL |
|
10× Capping Buffer |
0.1 mL |
0.4 mL |
2×1 mL |
2×10 mL |
2×100mL |
Storage
Store at -25 to -15°C. Valid for 2 years (avoid repeated freezing and thawing).
Storage buffer
20 mM Tris-HCl（pH 8.0，25℃), 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 0.1% Triton X-100，50% glycerol.
Activity Definition
One unit (U) of activity is defined as the amount of enzyme required to methylate 10 pmol of 80-nt capped RNA transcript in 1 hour at 37°C.
Quality Control
- Exonuclease activity: When 50 U of this product was reacted with 1 μg of λ-Hind III digested DNA at 37°C for 16 hours, the electrophoretic band of the DNA remained unchanged.
- Endonuclease activity: When 50 U of this product was reacted with 1 μg of λDNA at 37°C for 16 hours, the electrophoretic band of the DNA remained unchanged.
- Nickase activity: When 50 U of this product was reacted with 1 μg of pBR322 at 37°C for 16 hours, the electrophoretic band of the DNA remained unchanged.
- RNase activity: When 50 U of this product was reacted with 1.6 μg of MS2 RNA at 37°C for 4 hours, the electrophoretic band of the RNA remained unchanged.
- Residual E. coli DNA: Residual nucleic acid in 50 U of this product was detected by E. coli 16s rDNA-specific TaqMan qPCR, and the residual E. coli genome was ≤1 copy.
- Endotoxin: LAL-Test, Chinese Pharmacopoeia 2020 Edition Part IV Gel Limit Test General Rules (1143), bacterial endotoxin content ≤ 10 EU/mg.
Reaction system and conditions
- Dilute the appropriate amount of capped RNA to 16 μL with RNase-free water.
- Heat the diluted RNA at 65°C for 5 minutes, then place on ice for 5 minutes.
- Prepare the reaction mixture according to the following table (applicable to methylation reactions of up to 10 μg of capped RNA):
|
Components |
Volume |
|
Denatured capped RNA |
16 uL |
|
10× Capping Buffer* |
2 uL |
|
SAM (4 mM) |
1 uL |
|
mRNA Cap2'-O-Methyltransferase (50 U/μL) |
1 uL |
|
ddH2O |
Up to 20 uL |
*10× Capping Buffer: 500 mM Tris-HCl (pH 8.0, 25°C), 50 mM KCl, 10 mM MgCl₂, 10 mM DTT.
4, Incubate at 37°C for 1 hour (for RNA targets less than 200 nt in length, increase the incubation time to 2 hours).
Applications
Improve mRNA expression in microinjection and transfection experiments.
Notes
- Before starting the reaction, RNA must be purified and dissolved in nuclease-free water. All solutions must be free of EDTA and salt ions.
- Before the reaction, it is recommended to heat the sample RNA at 65°C for 5 minutes to remove secondary structures at the 5' end of the transcript. If the 5' end of the transcript is complex, the heating time can be extended to 10 minutes.
- SAM reagent is less stable at pH 7–8 and 37°C and should be prepared immediately before use. You can pre-calculate the required amount of SAM and dilute the 32 mM SAM stock solution to a 4 mM working solution before the reaction. To prevent SAM degradation, the working solution should be kept on ice.
- For your safety and well-being, please wear a lab coat and disposable gloves during this procedure.