Product Class: Polymerase

SP6 RNA Polymerase

recombinant unique buffer
Catalog #SizeConcentration
M0207S2,000 units20,000 units/ml
M0207L10,000 units20,000 units/ml

Description

Highlights

  • Isolated from a recombinant source
  • RNA probe preparation for hybridization
  • mRNA generation for in vitro translation systems
  • Supplied with 10X Reaction Buffer

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.

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
  • 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 and 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 40°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. 

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.

Supporting Documents

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

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.
  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?
  1. SP6 In Vitro Transcription Reaction Protocol (M0207)

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.