HiScribe™ T7 ARCA mRNA Kit

Description

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. By using a DNA template encoding a poly(A) tail, the HiScribe T7 ARCA mRNA Kit can be used to synthesize capped and tailed mRNAs. The cap structure is added to the mRNA by co-transcriptional incorporation of Anti-Reverse Cap Analog (ARCA) (NEB #S1411) using T7 RNA Polymerase. Poly(A) tail is incorporated during the transcription reaction. The kit also includes DNase I and LiCl for DNA template removal and quick mRNA purification.

Additionally, the kit is capable of partial incorporation of modified UTP and CTP (up to 50% each) without affecting the mRNA yield significantly. By using a DNA template encoding a poly(A) tail, capped and tailed modified mRNA can be synthesized in a single reaction in 30 minutes. 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 KitFigure 2. Overview of mRNA synthesis workflow with the HiScribe T7-ARCA mRNA Kit

Kit Components

The following reagents are supplied with this product:

Store at (°C)Concentration
ARCA/NTP Mix-202X
T7 RNA Polymerase Mix-20
DNase I (RNase-free)-202 units/μl
LiCl Solution-20
CLuc Control Template-200.25 μg/μl

Properties and Usage

Materials Required but not Supplied

  • DNA template
  • Thermocycler or 37°C incubator.
  • Nuclease-free water
  • Buffer- or water-saturated phenol:chloroform
  • Ethanol
  • 3 M Sodium acetate, pH 5.2
  • 5 M Ammonium acetate
  • Spin columns
  • Gels, running buffers and gel box
  • Equipment for RNA analysis

Storage Temperature

-20°C

FAQs

  1. HiScribe™ T7 ARCA mRNA Kit (with tailing) What is the difference between the HiScribe T7 ARCA mRNA Kit (NEB #E2065) and the HiScribe T7 ARCA mRNA Kit (with Tailing)(NEB #E2060)?
  2. I currently use MessageMAX™ T7 ARCA-capped Message Transcription Kit, which mRNA synthesis kit from NEB should I use?
  3. I currently use mMessage mMachine® T7 Ultra Transcription Kit, which mRNA synthesis kit from NEB should I use?
  4. Can modified nucleotides be used with the HiScribe T7 ARCA mRNA kits?
  5. What is the difference between the HiScribe T7 ARCA mRNA kits and the HiScribe T7 High Yield RNA Synthesis Kit (E2040) and HiScribe T7 Quick RNA Synthesis Kit (E2050)?

Protocols

  1. Standard mRNA Synthesis (E2065)
  2. mRNA Synthesis with Modified Nucleotides (E2065)
  3. mRNA Purification (E2065)
  4. Evaluation of Reaction Products (E2065)

Manuals

The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name these document files: manual[Catalog Number].

Safety Data Sheet

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.