Qiagen, P7180L, T7 RNA Polymerase (50,000 U)

50,000 U/mL of T7 RNA Polymerase and 10X T7 RNA Polymerase Buffer

Features

- DNA-dependent RNA polymerase
- High specificity for the T7 promoter
- Catalyzes the Mg 2+ dependent synthesis of RNA from rNTPs

Product Details

T7 RNA Polymerase provides 5ʹ→ 3ʹ RNA synthesis of RNA from a DNA template. T7 RNA polymerase requires its T7 double-stranded DNA promoter to initiate transcription, but it can transcribe RNA from both single-stranded and double-stranded DNA templates. The enzyme is supplied in 50 mM Tris-HCl, 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 50% glycerol and <0.1% Triton X-100; pH 7.9 at 25°C.

10X T7 RNA Polymerase Buffer (B7180) contains 400 mM Tris-HCl, 60 mM MgCl 2 , 100 mM DTT and 20 mM spermidine; pH 7.9 at 25°C.

Performance

Test: Units tested
Purity: n/a
Specific activity: n/a
Single-stranded exonuclease: 500 U
Double-stranded exonuclease: 500 U
Double-stranded endonuclease: 500 U
E. coli DNA contamination: 500 U
RNAse DNA contamination: 500 U

- Storage temperature: –25°C to –15°C
- Molecular weight: 98,855 Daltons

Principle

The recombinant enzyme protein is produced by a recombinant E. coli strain carrying the T7 RNA Polymerase gene. One unit is defined as the amount of enzyme that will incorporate 1 nmol of ATP into acid-precipitable material in 1 hour at 37°C.

Procedure

Components: Final Concentration
Nuclease-free Water: N/A
10X Reaction Buffer (B7180): 1X
rNTP: 500 µM each
Template DNA: 0.2-1 µg
T7 RNA polymerase (P7180L): 100 U

- Set up the following reaction mixture in a total volume of 20 μL:

Usage Instructions

Synthesis of non-labeled RNA

2. Incubate reaction mixture at 37°C for 1 hour.

References: 1. Chamberlin, M. et al. (1973) J. Biol. Chem., 248, 2235-2244, 2245-2250. 2. Chamberlin, M. et al. (1982) in The Enzymes, 3rd edition, ed. P. D. Boyer (Academic Press, New York), 15, 87-108. 3. Sambrook, J. et al. (1989) Cold Spring Harbor Laboratory Press, Molecular Cloning: A Laboratory Manual., (2nd ed.), 5.40-5.43.

Quality Control

Unit activity was measured using a twofold serial dilution method. Dilutions of the enzyme were made in 50% glycerol containing T7 RNA Polymerase storage solution and added to 50 µL reactions containing a T7 promoter-containing plasmid DNA, 1X T7 RNA Polymerase Buffer, 3H-ATP and 400 µM each ATP, GTP, CTP and UTP. Reactions were incubated for 10 minutes at 37°C, placed on ice, and analyzed using the method of Sambrook and Russell ( Molecular Cloning , v3, 2001, pp. A8.25-A8.26)

Protein concentration is determined by OD 280 absorbance.

Physical purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver-stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the band's mass corresponding to the protein of interest in the diluted sample.

Single-stranded exonuclease is determined in a 50 µL reaction containing a radiolabeled single-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C.

Double-stranded exonuclease activity is determined in a 50 µL reaction containing a radiolabeled double-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C.

Double-stranded endonuclease activity is determined in a 50 µL reaction containing 0.5 µg of plasmid DNA and 10 µL of enzyme solution incubated for 4 hours at 37°C.

E. coli contamination is evaluated using 5 µL replicate samples of enzyme solution that are denatured and screened in a TaqMan qPCR assay for the presence of contaminating E. coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus.

Non-specific RNAse contamination is assessed using the RNAse Alert kit (Integrated DNA Technologies), following the manufacturer’s guidelines.

Applications

- RNA synthesis from DNA template
- RNA probe preparation
- mRNA therapeutics

This product is available for molecular biology applications such as: