This kit is also available without tailing reagents.
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    E2060S HiScribe™ T7 ARCA mRNA Kit (with Tailing) $518.00
    $466.20
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    *On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca
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    E2040S HiScribe T7 High Yield RNA Synthesis Kit - 50 rxns $314.00
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    E2050S HiScribe T7 Quick High Yield RNA Synthesis Kit - 50 rxns $383.00
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    E2065S HiScribe™ T7 ARCA mRNA Kit $443.00
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    M0276L E.coli Poly (A) Polymerase - 500 units $398.00
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    M0276S E.coli Poly (A) Polymerase - 100 units $99.00
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    M0303L DNase I (RNase-Free) - 5,000 units $398.00
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    M0303S DNase I (RNase-Free) - 1,000 units $99.00
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    M0307L RNase Inhibitor, Human Placenta - 10,000 units $450.00
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    M0307S RNase Inhibitor, Human Placenta - 2,000 units $112.00
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    M0314L RNase Inhibitor, Murine - 15,000 units $398.00
    $358.20
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    M0314S RNase Inhibitor, Murine - 3,000 units $99.00
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    M0366S mRNA Cap2'-O-Methyltransferase - 2,000 units $81.00
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    M0493S Q5 Hot Start High-Fidelity DNA Polymerase - 100 units $166.00
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    M2080S Vaccinia Capping System - 400 units $195.00
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    N0362S ssRNA Ladder - 25 gel lanes $97.00
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    N0450L Ribonucleotide Solution Set - 50 umol of each $386.00
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    N0450S Ribonucleotide Solution Set - 10 umol of each $97.00
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    N0466L Ribonucleotide Solution Mix - 50 umol of each $403.00
    $362.70
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    N0466S Ribonucleotide Solution Mix - 10 umol of each $101.00
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    S1404L m7G(5')ppp(5')G RNA Cap Structure Analog - 5 umol $858.00
    $772.20
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    S1404S m7G(5')ppp(5')G RNA Cap Structure Analog - 1 umol $214.00
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    S1405L m7G(5')ppp(5')A RNA Cap Structure Analog - 5 umol $858.00
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    S1405S m7G(5')ppp(5')A RNA Cap Structure Analog - 1 umol $214.00
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    S1407L G(5')ppp(5') RNA Cap Structure Analog - 5 umol $665.00
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    S1407S G(5')ppp(5') RNA Cap Structure Analog - 1 umol $166.00
    $149.40
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    S1411L 3'-O-Me-m7G(5')ppp(5')G RNA Cap Structure Analog - 5 umol $824.00
    $741.60
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    S1411S 3'-O-Me-m7G(5')ppp(5')G RNA Cap Structure Analog - 1 umol $206.00
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    VIEW COMPANION PRODUCTS HIDE COMPANION PRODUCTS

    Product Introduction

    • Generate up to 25 μg of capped and tailed mRNA per reaction
    • mRNA capping, DNA removal, mRNA tailing and purification complete in 2 hours. Competitive products have more pipetting steps and separate components.
    • Enables partial incorporation of 5mCTP, Pseudo-UTP and other modified CTP and UTP
    • Ultra high-quality components ensure mRNA integrity
    • ARCA-based capping in correct orientation ensures high translation efficiency
    • Template removal and mRNA purification reagents included
    • HiScribe kits contain twice the number of reactions as competitive products
    • Also available without tailing reagents (NEB #E2065)

    Product Information

    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. 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)Figure 2. Overview of mRNA synthesis work flow with the HiScribe T7 ARCA mRNA Kit (with tailing)

    Kit Components

    The following reagents are supplied with this product:

    Store at (°C) Concentration
    Poly(A) Polymerase Reaction Buffer -20 10 X
    LiCl Solution -20 0 %
    T7 RNA Polymerase Mix -20 0 %
    DNase I (RNase-free) -20 2 units/μl
    E. coli Poly(A) Polymerase -20 0 %
    CLuc Control Template -20 0.25 μg/μl
    ARCA/NTP Mix -20 2 X
    Product Categories:
    RNA Synthesis
    Applications:
    RNA Modification,
    In vitro Synthesis (IVT)

    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

    Troubleshooting

    • Control Reaction
      The CLuc control template DNA is a linearized plasmid containing the Cypridina luciferase gene under the transcriptional control of the T7 promoter. The size of the runoff transcript is 1.6 kb. The control reaction should yield ≥ 15 μg RNA transcript in 30 minutes.

      If the control reaction is not working, there may be technical problems during reaction set up. Repeat the reaction by following the protocol carefully; take all precautions to avoid RNase contamination. Contact NEB for technical assistance.

      The control plasmid sequence can be found here. The CLuc control template is generated by linearizing the plasmid with restriction enzyme Xba I.

    • Low Yield of Full-length RNA
      If the transcription reaction with your template generates full-length RNA, but the yield is significantly lower than expected, it is possible that contaminants in the DNA template are inhibiting the RNA polymerase, or the DNA concentration may be incorrect. Alternatively, additional purification of DNA template may be required. Phenol:chloroform extraction is recommended.

    • Low Yield of Short Transcript
      High yields of short transcripts (< 0.3 kb) are achieved by extending incubation time and increasing the amount of template. Incubation of reactions up to 16 hours (overnight) or using up to 2 μg of template will help to achieve maximum yield.

    • RNA Transcript Smearing on Denaturing Gel
      If the RNA appears degraded (e.g., smeared) on denaturing agarose or polyacrylamide gel, the DNA template is likely contaminated with RNase. DNA templates contaminated with RNase can affect the length and yield of RNA synthesized (a smear below the expected transcript length). We recommend evaluating the plasmid DNA template with the RNase Contamination assay Kit (NEB #E3320). If the plasmid DNA template is contaminated with RNase, perform phenol:chloroform extraction, then ethanol precipitate and dissolve the DNA in nuclease-free water.

    • RNA Transcript of Larger Size than Expected
      If the RNA transcript appears larger than expected on a denaturing gel, plasmid DNA may be incompletely digested. Even small amounts of undigested circular plasmid DNA can produce large amounts of long transcripts. Check template for complete digestion. If undigested plasmid is confirmed, repeat restriction enzyme digestion.

      Larger size bands may also be observed when the RNA transcript is not completely denatured due to the presence of strong secondary structures.

    • RNA Transcript of Smaller Size than Expected
      If denaturing gel analysis shows the presence of smaller bands than the expected size, it is most likely due to premature termination by the polymerase. Sequences with resemblance to T7 RNA Polymerase termination signals will cause premature termination. Incubating the transcription reaction at lower temperatures, for example at 30°C, may increase the proportion of full-length transcript, however the yield will be decreased. For GC rich templates, or templates with secondary structures, incubation at 42°C may improve yield of full-length transcript.

    • Tailing Length Control
      A standard 30 min tailing reaction can add a poly(A) tail at least 150 nt in length to an average size mRNA generated from the IVT reaction. Short RNA may require longer incubation time for sufficient tailing.

    • No Tailing or Partial Tailing
      3´ end of the mRNA must be exposed for efficient tailing. Because T7 RNA Polymerase tends to generate 3´ end heterogenei

      Notes

      1. 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.

    Faqs & Tech Tips

    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 mMessage mMachine® T7 Ultra Transcription Kit, which mRNA synthesis kit from NEB should I use?
    3. I currently use MessageMAX™ T7 ARCA-capped Message 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)?
    6. Can I use the Monarch RNA Cleanup Kits to cleanup my in vitro transcription (IVT) reaction?

    Protocols & Manuals

    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].)

    Protocols

    1. Standard mRNA Synthesis (E2060)
    2. mRNA Synthesis with Modified Nucleotides (E2060)
    3. mRNA Purification (E2060)
    4. Evaluation of Reaction Products (E2060)

    Quality & Safety

    Quality Assurance Statement

    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.

    Specifications

    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]

    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]

    Safety DataSheets

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

    Poly(A) Polymerase Reaction Buffer
    LiCl Solution
    T7 RNA Polymerase Mix
    DNase I (RNase-free)
    E. coli Poly(A) Polymerase
    CLuc Control Template
    ARCA/NTP Mix