T7 RNA Polymerase - recombinant, expressed in E. coli

Catalog Number R0884
Storage Temperature -20 °C


CAS RN 9014-24-8
EC 2.7.7.6

Product Description

T7 RNA Polymerase is a DNA-dependant RNA Polymerase that exhibits a very high specificity for the T7 promoter sequence. The polymerase is useful for synthesizing large amounts of RNA suitable for in vitro translation and anti-sense RNA research. It can also produce highly sensitive radiolabeled RNA transcripts suitable for hybridization probes. There are many plasmid vectors available that contain a T7 promoter site upstream from a multiple cloning site. In addition, there are many plasmid and phage vectors that have a second promoter site either for SP6 or T3 RNA polymerase in the opposite orientation. With these vectors, sense or anti-sense transcripts can easily be obtained using the appropriate polymerase.

Molecular mass: 98.8 kDa

The product is supplied in a solution of 50% glycerol, 100 mM NaCl, 50 mM Tris-HCl, pH 7.9, 1 mM dithiothreitol, 0.1% w/v TRITON® X-100, and 0.1 mM EDTA.

Activity: 10,000-50,000 units/ml

Unit Definition: One unit of enzyme incorporates 1 nmole of nucleoside triphosphate into acid-insoluble material in 1 hour at 37 °C using activity assay conditions: 40 mM Tris-HCl, pH 7.9, 6 mM MgCl2, 2 mM spermidine, 0.5 mM each of ATP, CTP, and GTP, α-32P-UTP, 10 mM dithiothreitol, and 1 µg of DNA template.

DNase and RNase: None detected

Functional Assay: T7 RNA Polymerase is functionally tested and transcripts are analyzed by TCA precipitation, PAGE, and autoradiography.

Precautions and Disclaimer

This product is for R&D use only, not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.

Storage/Stability

Store at -20 °C.

Procedure

To obtain defined length transcripts from plasmid DNA, cleave the plasmid with the appropriate restriction enzyme. The length of the resulting transcript is defined by the promoter at its 5' end and the restriction site at its 3' end. Avoid choosing restriction enzymes that leave the template with a 3' overhang as this may result in incorrectly initiated transcripts. If a restriction site with a 3' overhang cannot be avoided, the template DNA ends should be "polished". Use either the Klenow fragment of DNA polymerase I to fill in or S1 nuclease to cleave the 3' overhang. S1 nuclease must be removed prior to running transcription reaction.

The yield of full length RNA transcripts is dependent on the ratio of DNA template to rNTPs. A typical 20 µl labeling reaction would include 1 µg of template DNA, 50 µCi α-32P-UTP or CTP at 400-800 Ci/mmole and a final concentration of 500 µM for the three complementary nucleotides. The limiting nucleotide concentration in a labeling reaction should be between 3 µM and 500 µM. When the concentration of the limiting nucleotide is below 3 µM, the incorporation efficiency is reduced and fewer full-length copies are generated. Avoid use of excessive T7 RNA polymerase as this may result in nonspecific initiations.

Note: RNase contamination must be avoided. The use of RNase free reagents, DNA template, and labware is essential. Including placental RNase inhibitor in the transcription reaction may be beneficial.

A. Labeled Transcription Reaction

The following conditions are typical of what would be used for producing a probe on a Northern blot to detect a sufficiently abundant mRNA.

  1. In a final volume of 20 µl, add 1 µg of template DNA in 40 mM Tris-HCl, pH 8, 8 mM MgCl2, 50 mM NaCl, 2 mM spermidine, 5 mM dithiothreitol, 500 µM each of ATP, CTP, and GTP, 50 µCi α-32P-UTP (800 Ci/mmol) with 10 units of T7 RNA polymerase.

  2. Incubate at 37 °C for 30 minutes. Stop by adding 2 µl of 0.2 M EDTA and/or heating to 65 °C.

  3. If necessary, unreacted nucleotides can be removed by gel filtration using the appropriate Sephadex® (Catalog Numbers S5772, S5897, S6022, S6147) or by using an RNA purification kit such as the GenElute™ Mammalian Total RNA Kit (Catalog Number RTN70).

B. Unlabeled Transcription Reaction

  1. If microgram quantities (up to 10 µg) of cold transcript RNA are desired, use the buffer conditions described in “Labeled Transcription Reaction” with the following exceptions: Use 2 µg of template DNA, 500 µM each of all 4 rNTPs, and 20 units of T7 RNA polymerase.

  2. Incubate at 37 °C for 60 minutes.

  3. If desired, the template DNA can be removed by the addition of 2 units of RNase free DNase I (Catalog Number D7291). Incubate for 15 minutes at 37 °C. Stop with 2 µl 0.2 M EDTA.

Materials


     

References

  1. Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1989) p. 10.27-10.37.
  2. Tabor, S., and Richardson, C.C., Proc. Natl. Acad. Sci. USA, 82, 1074-1078 (1985).
  3. Axelrod, V.D., and Kramer, F.R., Transcription from bacteriophage T7 and SP6 RNA polymerase promoters in the presence of 3'-deoxyribonucleoside 5¢-triphosphate chain terminators. Biochemistry, 24, 5716-5723 (1985).
  4. Chamberlin, M., and Ring, J., Characterization of T7-specific ribonucleic acid polymerase. 1. General properties of the enzymatic reaction and the template specificity of the enzyme. J. Biol. Chem., 248, 2235-2244 (1973).

 

GenElute is a trademark of Sigma-Aldrich® Co. LLC
TRITON is a trademark of The Dow Chemical Company or an affiliated company of Dow.
Sephadex is a registered trademark of GE Healthcare.

DXP,KTA,MAM 05/08-1

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