- Isolated from a recombinant source
- RNA probe preparation for hybridization
- mRNA generation for in vitro translation systems
- Supplied with 10X Reaction Buffer
SP6 RNA Polymerase catalyzes the synthesis of RNA in the 5´→ 3´ direction in the presence of a DNA template containing an SP6 phage promoter. SP6 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 run off transcription (1).
Product SourceAn E. coli strain that carries the cloned gene for SP6 RNA Polymerase from Salmonella typhimurium LT2Z.
The following reagents are supplied with this product:
|Store at (°C)||Concentration|
|RNAPol Reaction Buffer||-20||10X|
Advantages and Features
- Radiolabeled RNA probe preparation (2)
- RNA generation for in vitro translation (3)
- RNA generation for studies of RNA structure, processing and catalysis (3)
- Expression control via anti-sense RNA
Properties and Usage
Unit DefinitionOne 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 40°C in 1X RNA Polymerase Reaction Buffer.
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
10 mM DTT
2 mM spermidine
pH 7.9 @ 25°C
50 mM Tris-HCl
100 mM NaCl
20 mM β-ME
1 mM EDTA
0.1% Triton® X-100
pH 7.9 @ 25°C
Quality Assurance Statement
- Purified free of other RNA polymerases, DNases and RNases.
Quality Control AssaysThe 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.
- 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 (2 Hour Digestion):
The product is tested in a reaction containing a RNA substrate. After incubation for 2 hours there is no detectable degradation of the RNA substrate as determined by gel electrophoresis.
Material Safety Datasheets
- Dithiothreitol is required for activity.
- SP6 RNA Polymerase is extremely sensitive to salt inhibition. For best results, overall salt concentration should not exceed 50 mM.
- SP6 RNA Polymerase is 30% more active at 40°C than at 37°C
- 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.
- 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.
- Schenborn, E.T. and Meirendorf, R.C. (1985). Nucl. Acids Res. 13, 6223-6236.
- Butler, E.T. and Chamberlin, J. (1982). J. Biol. Chem. 257, 5772-5778.
- Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual . (2nd ed.), 10.27-10.37.
- Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. (2nd ed.), 18.82-18.84.
- Melton, D.A., Krieg, P.A., Rebagliati, M.R., Maniatis, T., Zinn, K. and Green, M.R. (1984). Nucl. Acids Res. 12, 7057-7070.
- Kreig, P.A. and Melton, D.A. (1984). Nucl. Acids Res. 12, 7057-7070.
- Green, M.R., Maniatis, R. and Melton, D.A. (1983). Cell. 32, 681-694.
- Melton, D.A. (1985). Proc. Natl. Acad. Sci. USA. 82, 144-128.
- Milligan, J.F., Groebe, D.R., Witherell, G.W. and Uhlenbeck, O.C. (1987). Nucl. Acids Res. 15, 8783.
- Zinn, K. et al. (1983). Cell. 34, 865-879.
- What is the molecular weight of SP6 RNA Polymerase?
- What are the main causes of reaction failure using SP6 RNA Polymerase?
- Is SP6 RNA Polymerase an enzyme of choice for making high specific activity labeled probes?
- Does the reaction with SP6 RNA Polymerase require a primer?
- What is the first base that SP6 RNA Polymerase transcribes?
- Does SP6 RNA Polymerase leave an extra base at the end of a transcript?
- Does SP6 RNA Polymerase require single stranded substrate?
- Will SP6 RNA Polymerase work on single stranded substrate?
- Will SP6 RNA Polymerase work on uncut plasmid DNA?
- Can aberrant RNA be produced when using SP6 RNA Polymerase?
- How can the yield of RNA be maximized when using SP6 RNA Polymerase?
- Why is the specific activity of the probe low?
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.