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  • T7 RNA Polymerase

    Did you know this product can be customized or purchased in larger volumes? Submit an inquiry to find out more.
    recombinant incubation temp
    Catalog #SizeConcentrationPriceQtyAdd to Cart
    M0251S5,000 units50,000 units/ml$66.00Add to Cart
    M0251L25,000 units50,000 units/ml$264.00Add to Cart
      
    Categories:
    RNA Synthesis Products

    Description

    Bacteriophage T7 RNA Polymerase is a DNA-dependent RNA polymerase that is highly specific for the T7 phage promoters. The 99 KD enzyme catalyzes in vitro RNA synthesis from a cloned DNA sequence under the T7 promoters. RNA produced using the T7 RNA Polymerase is suitable for many applications in research and biotechnology. 

    Reaction Conditions
    1X RNAPol Reaction Buffer, supplemented with 0.5 mM each ATP, UTP, GTP, CTP, and DNA template containing the T7 RNA Polymerase promoter. Incubate at 37°C. 

    Highlights

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

    Product Source

    Isolated from E.coli BL21 carrying the plasmid pAR1219 which contains T7 gene I. 

    Reagents Supplied

    The following reagents are supplied with this product:

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

    Advantages and Features

    Applications

    • Radiolabeled RNA probes
    • Non-isotopic RNA labeling
    • Preparation of RNA vaccines
    • Guide RNA for gene targeting
    • mRNA for in vitro translation and micro injection 
    • RNA structure, processing and catalysis studies
    • RNA amplification
    • Anti-sense RNA for gene expression experiment 

    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.

    1X RNAPol Reaction Buffer
    40 mM Tris-HCl
    6 mM MgCl2
    2 mM spermidine
    1 mM dithiothreitol
    pH 7.9 @ 25°C

    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

    +++

    Unit Assay Conditions

    1X RNAPol Reaction Buffer, supplemented with 0.5 mM each ATP, UTP, GTP, CTP, and 1 µg T7 DNA in 50 µl.

    Notes

    1. For radio labeled high specific activity RNA probes, the concentration of the radioactive nucleotide should be limited to 6 μM. 
    2. To protect RNA against ribonuclease, RNase inhibitor (NEB #M0314 or M#0307) should be added to a final concentration of 1 U/ μl. 
    3. T7 RNA Polymerase is extremely sensitive to salt inhibition. The overall salt concentration should not exceed 50 mM. 

    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.

    FAQs

    1. What is the promoter sequence of T7 RNA Polymerase?
    2. Is it possible to start transcription with an A?
    3. Does the transcription reaction with T7 RNA Polymerase require a primer?
    4. Does T7 RNA Polymerase leave an extra base at the end of a transcript?
    5. Will T7 RNA Polymerase work on single stranded substrate?
    6. Will T7 RNA Polymerase work on uncut plasmid DNA?
    7. Can aberrant RNA be produced when using T7 RNA Polymerase?
    8. How can the yield of RNA be maximized when using T7 RNA Polymerase?
    9. Can I use T7 RNA Polymerase to make high specific activity radiolabeled probes?
    10. Why is the specific activity of the probe low?
    11. What are the main causes of reaction failure using T7 RNA Polymerase?

    Protocols

    1. Protocol for Standard RNA Synthesis

    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.

    Selection Tools

    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.
    • 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.
    • Protein Purity (SDS-PAGE):
      The physical purity is assessed by comparing contaminating protein bands in a concentrated sample to the protein of interest band in a sample of known dilution. The purity is determined by SDS-PAGE.
    • 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 (Extended Digestion):
      The product is tested in a reaction containing a RNA substrate. After incubation for 16 hours greater than 90% of the substrate RNA remains intact as determined by gel electrophoresis.

    Certificate of Analysis

    The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality control's for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

    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]

    Safety Data Sheet

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

    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.