- Isolated from a recombinant source
- RNA probe preparation for hybridization
- mRNA generation for in vitro translation systems
1X RNAPol Reaction Buffer, supplemented with 0.5 mM each ATP, UTP, GTP, CTP, and DNA template containing the T7 RNA Polymerase promoter. Incubate at 37°C.
Product SourceIsolated from E.coli BL21 carrying the plasmid pAR1219 which contains T7 gene I.
The following reagents are supplied with this product:
|Store at (°C)||Concentration|
|RNAPol Reaction Buffer||-20||10X|
Advantages and Features
- Radiolabeled RNA probes
- Non-isotopic RNA labeling
- Preparation of 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 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.
1X RNAPol Reaction Buffer
40 mM Tris-HCl
6 mM MgCl2
2 mM spermidine
1 mM dithiothreitol
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
Unit Assay Conditions1X RNAPol Reaction Buffer, supplemented with 0.5 mM each ATP, UTP, GTP, CTP, and 1 µg T7 DNA in 50 µl.
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.
- 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.
- For radio labeled high specific activity RNA probes, the concentration of the radioactive nucleotide should be limited to 6 μM.
- To protect RNA against ribonuclease, RNase inhibitor (NEB #M0314 or M#0307) should be added to a final concentration of 1 U/ μl.
- T7 RNA Polymerase is extremely sensitive to salt inhibition. The overall salt concentration should not exceed 50 mM.
- 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?