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  • T7 RNA Polymerase

    Description

    T7 RNA Polymerase catalyzes the synthesis of RNA in the 5´→ 3´ direction in the presence of a DNA template containing a T7 phage promoter. T7 RNA Polymerase can be used to generate high specific activity labeled RNA probes, RNA for in vitro translation, biologically active mRNA, and/or preparative quantities of defined length RNA by the run off transcription method (1).

    Highlights

    • Isolated from a recombinant source
    • RNA probe preparation for hybridization
    • mRNA generation for in vitro translation systems

    Product Source

    An E. coli strain containing a recombinant gene encoding T7 RNA Polymerase.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    RNAPol Reaction Buffer-2010X

    Advantages and Features

    Applications

    • Radiolabeled RNA probe preparation
    • RNA generation for in vitro translation
    • RNA generation for studies of RNA structure, processing and catalysis
    • Expression control via anti-sense RNA

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme that will incorporate 1 nmol ATP into acid-insoluble material in a total reaction volume of 50 μl in 1 hour at 37°C in 1X RNA Polymerase Reaction Buffer.

    Storage Conditions

    50 mM Tris-HCl
    100 mM NaCl
    20 mM β-ME
    1 mM EDTA
    50% Glycerol
    0.1% Triton® X-100
    pH 7.9 @ 25°C

    Molecular Weight

    Theoretical: 98000 daltons

    Specific Activity

    1,200,000 units/mg

    5' - 3' Exonuclease

    No

    3' - 5' Exonuclease

    No

    Strand Displacement

    +++

    Quality Control

    Quality Assurance Statement

    • Purified free of other RNA polymerases, DNases and RNases.

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Endonuclease Activity (Nicking):
      The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
    • Exonuclease Activity (Radioactivity Release):
      The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
    • Ligation and Recutting (Terminal Integrity, Digested DNA):
      The product is tested in a reaction containing DNA fragments with known ends, the percentage of the DNA fragments ligated with T4 DNA ligase and the percentage that can be recut are determined by agarose gel electrophoresis.
    • Non-Specific DNase Activity (16 hour):
      The product is tested for non-specific nuclease degradation in a reaction containing a DNA substrate. After incubation for 16 hours there is no detectable degradation of the DNA substrate as determined by agarose gel electrophoresis.
    • RNA Polymerase Specificity:
      The RNA Polymerase is tested for non-specific RNA Polymerase activity using Lambda DNA as the template. Lambda DNA does not contain the appropriate promoter sequence.
    • RNase Activity (1 Hour Digestion):
      The product is tested in a reaction containing a RNA substrate.  After incubation for 1 hour there is no detectable degradation of the RNA substrate as determined by gel electrophoresis.

    Notes

    1. Dithiothreitol is required for activity.
    2. T7 RNA Polymerase is extremely sensitive to salt inhibition.
    3. For best overall salt concentration should not exceed 50 mM.
    4. Higher yields of RNA may be obtained by raising NTP concentrations (up to 4 mM each). Mg2+ concentration should be raised to 4 mM above the total NTP concentration. Additionally, inorganic pyrophosphatase should be added to a final concentration of 4 units/ml.
    5. An apparent decrease in enzyme activity over time may be due to the breakdown of dithiothreitol in the reaction buffer; even when stored at -20°C. If you observe a decrease in yield, try supplementing your reactions with a final concentration of 10 mM fresh dithiothreitol.

    References

    1. Schenborn, E.T. and Meirendorf, R.C. (1985). Nucl. Acids Res.. 13, 6223-6236.
    2. Davanloo, P., Rosenberg, A.H., Dunn, J.J. and Studier, F.W. (1984). Proc. Natl. Acad. Sci. USA. 81, 2035-2039.
    3. Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, (2nd Ed.). 10.27-10.37.
    4. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, (2nd Ed.). 18.82-18.84.
    5. Melton, D.A., Kreig, P.A., Rebagliati, M.R., Maniatis, T., Zinn, K. and Green, M.R. (1984). Nucl. Acids Res.. 12, 7035-7056.
    6. Milligan, J.F., Groebe, D.R., Witherell, G.W. and Uhlenbeck, O.C. (1987). Nucl. Acids Res.. 15, 8783.
    7. Noren, C.J. et al. (1990). Nucl. Acids Res.. 18, 83-88.
    8. Kreig, P.A. and Melton, D.A. (1984). Nucl. Acids Res.. 12, 7057-7070.

    Supporting Documents

    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]

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Datacards

    The Product Summary Sheet, or Data Card, 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 the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. What are the main causes of reaction failure using T7 RNA Polymerase?
    2. Is T7 RNA Polymerase an enzyme of choice for making high specific activity labeled probes?
    3. Does the reaction with T7 RNA Polymerase require a primer?
    4. What is the first base that T7 RNA Polymerase transcribes?
    5. Does T7 RNA Polymerase leave an extra base at the end of a transcript?
    6. Does T7 RNA Polymerase require single stranded substrate?
    7. Will T7 RNA Polymerase work on single stranded substrate?
    8. Will T7 RNA Polymerase work on uncut plasmid DNA?
    9. Can aberrant RNA be produced when using T7 RNA Polymerase?
    10. How can the yield of RNA be maximized when using T7 RNA Polymerase?
    11. Why is the specific activity of the probe low?

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