- Isolated from a recombinant source
- RNA probe preparation for hybridization
- mRNA generation for in vitro translation systems
Product SourceAn E. coli strain containing a recombinant gene encoding T7 RNA Polymerase.
The following reagents are supplied with this product:
|Store at (°C)||Concentration|
|RNAPol Reaction Buffer||-20||10X|
Advantages and Features
- Radiolabeled RNA probe preparation
- RNA generation for in vitro translation
- RNA generation for studies of RNA structure, processing and catalysis
- 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 37°C in 1X RNA Polymerase Reaction Buffer.
1X RNAPol Reaction Buffer
40 mM Tris-HCl
6 mM MgCl2
1 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
Molecular WeightTheoretical: 98000 daltons
Specific Activity1,200,000 units/mg
5' - 3' ExonucleaseNo
3' - 5' ExonucleaseNo
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.
- Ligation and Recutting (Terminal Integrity, Digested DNA):
The product is tested in a reaction containing DNA fragments with known ends, the percentage of the DNA fragments ligated with T4 DNA ligase and the percentage that can be recut are determined by agarose gel electrophoresis.
- 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 (1 Hour Digestion):
The product is tested in a reaction containing a RNA substrate. After incubation for 1 hour there is no detectable degradation of the RNA substrate as determined by gel electrophoresis.
- Dithiothreitol is required for activity.
- T7 RNA Polymerase is extremely sensitive to salt inhibition.
- For best overall salt concentration should not exceed 50 mM.
- 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.
- Davanloo, P., Rosenberg, A.H., Dunn, J.J. and Studier, F.W. (1984). Proc. Natl. Acad. Sci. USA. 81, 2035-2039.
- Sambrook, J., Fritsch, E.F. and Maniatis, T. 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., Kreig, P.A., Rebagliati, M.R., Maniatis, T., Zinn, K. and Green, M.R. (1984). Nucl. Acids Res.. 12, 7035-7056.
- Milligan, J.F., Groebe, D.R., Witherell, G.W. and Uhlenbeck, O.C. (1987). Nucl. Acids Res.. 15, 8783.
- Noren, C.J. et al. (1990). Nucl. Acids Res.. 18, 83-88.
- Kreig, P.A. and Melton, D.A. (1984). Nucl. Acids Res.. 12, 7057-7070.
- What is the promoter sequence of T7 RNA Polymerase?
- Is it possible to start transcription with an A?
- Does the transcription reaction with T7 RNA Polymerase require a primer?
- Does T7 RNA Polymerase leave an extra base at the end of a transcript?
- Will T7 RNA Polymerase work on single stranded substrate?
- Will T7 RNA Polymerase work on uncut plasmid DNA?
- Can aberrant RNA be produced when using T7 RNA Polymerase?
- How can the yield of RNA be maximized when using T7 RNA Polymerase?
- Can I use T7 RNA Polymerase to make high specific activity radiolabeled probes?
- Why is the specific activity of the probe low?
- What are the main causes of reaction failure using T7 RNA Polymerase?