Cat. No. / ID: P7180L
T7 RNA Polymerase provides 5ʹ→ 3ʹ RNA synthesis of RNA from a DNA template.
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 the following: 400 mM Tris-HCl, 60 mM MgCl2, 100 mM DTT and 20 mM spermidine; pH 7.9 at 25°C.
T7 RNA Polymerase is a DNA-dependent RNA polymerase having high specificity for the T7 promoter. After promoter initiation, it catalyzes the Mg2+ dependent synthesis of RNA from rNTPs.
|Specific activity||312,500 U/mg|
|Single-stranded exonuclease||500 U||<1.0% released|
|Double-stranded exonuclease||500 U||<1.0% released|
|Double-stranded endonuclease||500 U||No conversion|
|E. coli DNA contamination||500 U||<10 copies|
|RNAse comtamination||500 U||No detectable non-specific RNAse|
Source of recombinant enzyme protein
The protein is produced by a strain ofE. coli that expresses the recombinant T7 RNA Polymerase gene.
Unit definition: 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.
Molecular weight: 98,855 Daltons
Quality control analysis
Unit activity was measured using a twofold serial dilution method. Dilutions of 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 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 OD280 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 mass of the band 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.
This OEM by QIAGEN product is available for bulk purchase for the following commercial assay applications.