Product Class: Other

SP6 RNA Polymerase
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Item# Description List Price Web Price Qty
M0207L SP6 RNA Polymerase - 10,000 units $390.00
$351.00
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M0207S SP6 RNA Polymerase - 2,000 units $97.00
$87.30
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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

The large size of this product will be discontinued on 12/31/2019. The small size will still be available.

Product Introduction

SP6 RNA Polymerase is used for in vitro mRNA synthesis and is highly specific for the SP6 promoter.

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

Description

Bacteriophage SP6 RNA Polymerase is a DNA-dependent RNA polymerase that is highly specific for the SP6 phage promoter. The 98.5 KD polymerase catalyzes in vitro RNA synthesis from a cloned DNA template under the SP6 promoter. RNA synthesized using the SP6 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 SP6 RNA Polymerase promoter. Incubate at 37°C. 

Product Source

An E. coli strain that carries the cloned gene for SP6 RNA Polymerase from Salmonella typhimurium LT2Z.

Advantages and Features

Application Features

  • Radiolabeled RNA probe preparation
  • Non-isotopic RNA labeling
  • Preparation for 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 & Usage

    Unit Definition

    One unit is defined as the amount of enzyme required to incorporate 1 nmol ATP into an acid-insoluble material in 1 hour at 37°C. 

    Reaction Conditions

    1X RNAPol Reaction Buffer
    Supplement with 0.5 mM GTP, 0.5 mM ATP , 0.5 mM UTP  and 0.5 mM CTP 
    Incubate at 37°C

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

    Storage Temperature

    -20°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

    Unit Assay Conditions

    1X RNAPol Reaction Buffer, supplemented with 0.5 mM each ATP, UTP, GTP, CTP, and 1 μg DNA containing the SP6 promoter in 50 μl. 

    Product 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 #M0307) should be added to a final concentration of 1 U/μl. 
    3. SP6 RNA Polymerase is extremely sensitive to salt inhibition. The overall salt concentration should not exceed 50 mM. 
    4. SP6 RNA Polymerase is slightly more active at 40°C than at 37°C. Incubation at 40°C may be considered for RNA transcript containing strong secondary structures. 

    References

    1. Schenborn, E.T. and Meirendorf, R.C. (1985). Nucl. Acids Res. 13, 6223-6236.
    2. Butler, E.T. and Chamberlin, J. (1982). J. Biol. Chem. 257, 5772-5778.
    3. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). 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., Krieg, P.A., Rebagliati, M.R., Maniatis, T., Zinn, K. and Green, M.R. (1984). Nucl. Acids Res. 12, 7057-7070.
    6. Kreig, P.A. and Melton, D.A. (1984). Nucl. Acids Res. 12, 7057-7070.
    7. Green, M.R., Maniatis, R. and Melton, D.A. (1983). Cell. 32, 681-694.
    8. Melton, D.A. (1985). Proc. Natl. Acad. Sci. USA. 82, 144-128.
    9. Milligan, J.F., Groebe, D.R., Witherell, G.W. and Uhlenbeck, O.C. (1987). Nucl. Acids Res. 15, 8783.
    10. Zinn, K. et al. (1983). Cell. 34, 865-879.

    Protocols, Manuals & Usage

    Protocols

    1. SP6 In Vitro Transcription Reaction Protocol (M0207)

    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 [email protected] or fill out the Technical Support Form for appropriate document.)

    Tools & Resources

    Selection Charts

    • DNA Polymerase Selection Chart
    • RNA Polymerase Selection Chart

    FAQs & Troubleshooting

    FAQs

    1. Does the transcription reaction with SP6 RNA Polymerase require a primer?
    2. Does SP6 RNA Polymerase leave an extra base at the end of a transcript?
    3. Will SP6 RNA Polymerase work on single stranded substrate?
    4. Will SP6 RNA Polymerase work on uncut plasmid DNA?
    5. Can aberrant RNA be produced when using SP6 RNA Polymerase?
    6. Can I use SP6 RNA Polymerase to make high specific activity radiolabeled probes?
    7. Why is the specific activity of the probe low?
    8. What are the main causes of reaction failure using SP6 RNA Polymerase?

    Troubleshooting

    Tech Tips

    Supplement reactions with fresh DTT if buffer is > 6 months old. 

    Increase yields by raising rNTP concentrations to 4 mM each and MgCl2 to 20 mM.

    Quality, Safety & Legal

    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.

    Legal and Disclaimers

    This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).

    While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.

    For more information about commercial rights, please contact NEB's Global Business Development team at [email protected].

    This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

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