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  • HiScribe™ T7 Quick High Yield RNA Synthesis Kit

    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
    NTP Buffer Mix-2020 mM of each NTP
    T7 RNA Polymerase Mix-20
    FLuc Control Template-200.5 μg/μl
    DNase I (RNase-free)-202 units/μl
    LiCl Solution-20

    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

    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 or use of single-stranded DNA binding protein (SSB) may improve yield of full-length transcript.

    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 (NEB #M0386)

    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].

    Interactive Tools

    Safety Data Sheet

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