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.
Source:
Escherichia coli
Purity:
>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.
Storage:
-20°C or -80°C for 12 months under sterile conditions from date of receipt.
Notes:
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.
Shipping Conditions:
Dry ice
Escherichia coli
Purity:
>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.
Storage:
-20°C or -80°C for 12 months under sterile conditions from date of receipt.
Notes:
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.
Shipping Conditions:
Dry ice
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:
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).
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 |
5. Incubate at 37°C for 60 minutes (For RNA less than 200 nt long increase incubation time to 2 hours).
