Product Class: Other

SP6 RNA Polymerase
NEBU recombinant 37

Product Introduction

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

Catalog # Size Concentration
M0207S 2000.0 units 20000 units/ml

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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.
This product is related to the following categories:
cDNA Synthesis & Reverse Transcriptases Products,
RNA Synthesis In vitro Transcription (IVT),
This product can be used in the following applications:
PCR

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • M0207S     -20    
      SP6 RNA Polymerase M0207SVIAL -20 1 x 0.1 ml 20,000 units/ml
      RNAPol Reaction Buffer B9012SVIAL -20 1 x 1 ml 10 X

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 ATP, 0.5 mM GTP, 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 Buffer

50 mM Tris-HCl
100 mM NaCl
20 mM β-ME
1 mM EDTA
50% Glycerol
0.1% (w/v) 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. 

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

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. Zinn, K. et al. (1983). Cell. 34, 865-879.
  3. Butler, E.T. and Chamberlin, J. (1982). J. Biol. Chem. 257, 5772-5778.
  4. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual . (2nd ed.), 10.27-10.37.
  5. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. (2nd ed.), 18.82-18.84.
  6. Melton, D.A., Krieg, P.A., Rebagliati, M.R., Maniatis, T., Zinn, K. and Green, M.R. (1984). Nucl. Acids Res. 12, 7057-7070.
  7. Kreig, P.A. and Melton, D.A. (1984). Nucl. Acids Res. 12, 7057-7070.
  8. Green, M.R., Maniatis, R. and Melton, D.A. (1983). Cell. 32, 681-694.
  9. Melton, D.A. (1985). Proc. Natl. Acad. Sci. USA. 82, 144-128.
  10. Milligan, J.F., Groebe, D.R., Witherell, G.W. and Uhlenbeck, O.C. (1987). Nucl. Acids Res. 15, 8783.

Protocols, Manuals & Usage

Protocols

  1. SP6 In Vitro Transcription Reaction Protocol (M0207)

Application Notes

Tools & Resources

Selection Charts

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?

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

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

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.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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