Product Description:
The ScriptCap™ m7G Capping System adds a methylated guanine nucleotide cap to the 5′ end of RNA, generating Cap 0-RNA with ~100% efficiency. The process consists of three enzymatic reactions: (1) conversion of the 5′ triphosphate of RNA to a diphosphate, (2) joining of GTP to the 5′ diphosphate of the first nucleotide and (3) methylation of the 7-nitrogen of guanine using S-adenosyl-methionine (SAM). These reactions produce Cap 0-RNA with nearly 100% efficiency, a level that cannot be obtained using co-transcriptional capping methods. A standard reaction caps approximately 60 μg of RNA and can be scaled up or down to accommodate user needs.
5’ Capping enhances mRNA stability and translation efficiency in cells compared to uncapped RNA. Cap 0-RNA can be converted to Cap 1-RNA using the ScriptCap™ 2′-O-Methyltransferase Kit to further boost in vivo translation efficiency. ScriptCap™ capped RNA can be added directly to A-Plus™ Poly(A) Polymerase tailing reactions without cleanup for seamless synthesis of fully 5’ capped and 3’ tailed mRNA.
Materials Supplied:
Important Store at -20°C in a freezer without a defrost cycle. Do not store at -70°C.
ScriptCap™ m7G Capping System Contents (25 reactions)
|
Kit Component |
Reagent Volume |
ScriptCap™ Capping Enzyme, 10 U/μl
in 50% glycerol, 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT), 0.1 mM EDTA and 0.1% Triton® X-100 |
100 μl |
10X ScriptCap™ Capping Buffer
0.5 M Tris-HCl, pH 8.0, 60 mM KCl and 12.5 mM MgCl2 |
250 μl |
10 mM GTP |
250 μl |
20 mM S-adenosyl-methionine (SAM) |
30 μl |
ScriptGuard™ RNase Inhibitor, 40 U/μl
in 50% glycerol, 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM DTT, 0.1 mM EDTA and 0.1% Triton X-100 |
65 μl |
RNase-Free Water |
2 x 1.4 ml |
Materials Required, but not Supplied
- IVT RNA
- Materials or kits for purification of the RNA product
Terms & Conditions: The Product listed is currently offered by CELLSCRIPT™ for research use only under the defined Terms and Conditions.
FAQs: For answers to Frequently Asked Questions about our products, visit the FAQ library.
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Troubleshooting Guide:
Symptom |
Solution |
Low capping efficiency |
RNA to be treated with ScriptCap™ Capping Enzyme should be purified and resuspended in RNase-free water. Do not resuspend the RNA in an EDTA-containing solution. |
Prior to capping, purify the input RNA using a method that removes residual proteins, contaminants and unincorporated nucleotides. |
Verify that ScriptGuard™ RNase Inhibitor was added to the reaction. |
SAM slowly degrades at room temperature and above. Keep SAM solutions on ice at all times. Be sure to use fresh dilutions of SAM which have always been stored on ice. Inefficient N7 methylation will result in inefficient capping. |
Increase the RNA heat-denaturation conditions used. For example, 65°C for 20 minutes, 75°C for 10 minutes, 85°C for 5 minutes, etc. |
Increase the capping reaction incubation time. For example, up to 3 hours at 37°C. |
Some RNAs form stable structures (e.g., homodimers, hairpins) at the 5' end, limiting access by Capping Enzyme. Analyze the sequence and increase the RNA denaturation temperature to above the Tm (e.g., to 65°C for 20 min, 75°C for 10 min, 85°C for 5 min). If the 5' end is highly structured, it might be necessary to modify the 5' end sequence using molecular biology techniques. Often this can be accomplished by making a single point mutation within the first 5 bases of the DNA template for the RNA transcript (non-coding region). |
White precipitate in reaction buffer |
Incubate the reaction buffer at 37°C for 5 minutes then mix thoroughly to dissolve the precipitate. |
Do not store the kit at –70°C. |
