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    E2050S HiScribe T7 Quick High Yield RNA Synthesis Kit - 50 rxns $387.00
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    Product Introduction

    • Up to 180 μg of RNA generated per reaction, from 1 μg of control template
    • Master mix format reduces pipetting steps while still enabling partial substitution of NTPs for labeling and incorporation of modified bases
    • Template removal and RNA purification reagents included
    • Linearized control template included for verification of RNA synthesis

    Product Information

    Description

    The HiScribe T7 Quick High Yield RNA Synthesis Kit is designed for quick set-up and production of large amounts of RNA in vitro. The reaction can be set up conveniently by combining the NTP buffer mix, T7 RNA Polymerase mix and a suitable DNA template. The kit also allows for capped RNA or dye-labeled RNA synthesis by incorporation of cap analog (ARCA, NEB #S1411) or dye-modified nucleotides. RNA synthesized with the kit can be used for RNA structure and function studies, ribozyme biochemistry, as probes for RNase protection assays and hybridization based blots, anti-sense RNA and RNAi experiments, microarray analysis and microinjection, as well as in vitro translation and RNA vaccines.

    To synthesize high specific activity radioactive RNA probes or RNA with 100% substitution of one or more modified nucleotides we recommend using the T7 High Yield RNA synthesis kit (NEB #E2040), in which the four nucleotides are supplied separately.

    DNA Template Preparation:
    Linearized plasmid DNA, PCR products or synthetic DNA oligonucleotides can be used as templates for in vitro transcription with the HiScribe T7 Quick High Yield RNA Synthesis Kit, provided that they contain a double-stranded T7 promoter region upstream of the sequence to be transcribed. Figure 1 illustrates the minimal T7 promoter sequence, as well as a run-off transcript after T7 transcription.

    Figure 1. Transcription by T7 RNA PolymeraseFigure 1. Transcription by T7 RNA Polymerase

    Kit Components

    The following reagents are supplied with this product:

    Store at (°C) Concentration
    LiCl Solution -20 0 %
    T7 RNA Polymerase Mix -20 0 %
    DNase I (RNase-free) -20 2 units/μl
    FLuc Control Template -20 0.5 μg/μl
    NTP Buffer Mix -20 20 mM of each NTP
    Product Categories:
    RNA Synthesis Products
    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

    Storage Temperature

    -20°C

    Troubleshooting


    • Control Reaction
      The FLuc control template DNA is a linearized plasmid containing the firefly luciferase gene under the transcriptional control of the T7 promoter. The size of the runoff transcript is 1.8 kb. The control reaction should yield ≥ 150 μg RNA transcript in 2 hours.

      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 FLuc control template is generated by linearizing the plasmid with StuI.

    • 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 (see template DNA preparation section).

    • 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) before plasmid DNA template is used in the HiScribe T7 Quick High Yield RNA Synthesis Kit. If the plasmid DNA template is contaminated with RNase, perform phenol:chloroform extraction, then ethanol precipitate and dissolve the DNA in nuclease-free water (see template DNA preparation section).

    • RNA Transcript of Larger Size than Expected
      If the RNA transcript appears larger than expected on a denaturing gel, template plasmid DNA may be incompletely digested. Even small amounts of undigested circular 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. Some 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.

    Faqs & Tech Tips

    FAQs

    1. 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.

    Protocols

    1. Standard RNA Synthesis (E2050)
    2. Capped RNA Synthesis (E2050)
    3. RNA Synthesis with Modified Nucleotides (E2050)
    4. Purification of Synthesized RNA (E2050)
    5. Evaluation of Reaction Products (E2050)
    6. In vitro digestion of DNA with Cas9 Nuclease, S. pyogenes (M0386)
    7. Poly(A) Tailing of RNA using E. coli Poly(A) Polymerase (NEB# M0276)
    8. Using recombinant Cas9 nuclease to assess locus modification in genome editing experiments (#M0386)
    9. sgRNA Synthesis Using the HiScribe™ Quick T7 High Yield RNA Synthesis Kit (NEB #E2050)

    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.

    LiCl Solution
    T7 RNA Polymerase Mix
    DNase I (RNase-free)
    FLuc Control Template
    NTP Buffer Mix