Most eukaryotic mRNAs require a 7-methyl guanosine (m7G) cap structure at the 5´ end and a poly(A) tail at the 3´ end to be efficiently translated. The HiScribe T7 ARCA mRNA Kit (with tailing) is designed for quick production of ARCA capped and poly(A) tailed mRNA in vitro. Capped mRNAs are synthesized by co-transcriptional incorporation of Anti-Reverse Cap Analog (ARCA, NEB #S1411) using T7 RNA Polymerase. The transcription reaction can be set up easily by combining the ARCA/NTP mix, T7 RNA Polymerase Mix and a suitable DNA template. The kit also allows for partial incorporation of 5mCTP, Pseudo-UTP and other modified nucleotides into mRNA. After a brief DNase I treatment to remove the template DNA, capped mRNA is poly(A) tailed with Poly(A) Polymerase. mRNAs synthesized with the kit can be used for cell transfection, microinjection, in vitro translation and RNA vaccines.
ARCA is incorporated into mRNA exclusively in the correct orientation, generating capped mRNA that is more efficiently translated. Standard cap analogs can be incorporated in either direction resulting in only 50% of capped mRNA that is functional in protein translation.
Figure 1. Structure of Anti-Reverse Cap Analog (ARCA, NEB #S1411)
Methylation at the 3´ position of 7mG forces the cap structure to be attached to mRNA in the correct orientation.Figure 2. Overview of mRNA synthesis work flow with the HiScribe T7 ARCA mRNA Kit (with tailing)
Properties & Usage
Materials Required but not Supplied
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DNA template
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Thermocycler or 37°C incubator.
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Nuclease-free water
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Buffer- or water-saturated phenol:chloroform
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Ethanol
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3 M Sodium acetate, pH 5.2
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5 M Ammonium acetate
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Spin columns (see Monarch® RNA Cleanup Kits, NEB #T2040 or #T2050)
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Gels, running buffers and gel box
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Equipment for RNA analysis
Related Products
Product Notes
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All kit components should be stored at -20°C. The kit contains sufficient reagents for 20 reactions of 20 μl each. Each standard reaction yields up to 20 μg of capped mRNA from 1 μg control template. Up to 25 μg capped and tailed mRNA can be obtained after Poly(A) tailing and purification by LiCl precipitation.
Protocols
Manuals
The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.
Quality Control Assays
Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.
Certificate of Analysis
The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
Specifications & Change Notifications
The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
The following link is to a PDF Safety Data Sheet (SDS) that applies to this product to help you use it safely.
Legal and Disclaimers
Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
New England Biolabs (NEB) is committed to practicing ethical science - we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.