Description:
T7 RNA Polymerase is a DNA-dependent RNA polymerase derived from the T7 bacteriophage which exhibits a high recognition specificity to the T7 promoter and terminator sequences and catalyzes the 5′→ 3′ synthesis of RNA starting at a T7 promoter sequence(1,2).
Specific Activity:
312,500U/mg
Applications:
- 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
Source:
Purified from a strain of E. coli that expresses the recombinant T7 RNA Polymerase gene.
Supplied in:
50 mM Tris-HCl
100 mM NaCl
1 mM dithiothreitol
1 mM EDTA
50% glycerol
0.1% Triton X-100
pH 7.9 @ 25°C
Supplied with:
10X T7 RNA Polymerase Buffer
400 mM Tris-HCl
60 mM MgCl2
100 mM Dithiothreitol
20 mM Spermidine
pH 7.9 @ 25°C
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.
Nuclease Contamination Tests
Endonuclease Activity:
A 50 µL reaction containing 1 µg of pBR322 DNA and 10 µL of enzyme solution incubated for 4 hours at 37°C resulted in no visually discernible conversion to nicked circular DNA as determined by agarose gel electrophoresis.
Single-Stranded Exonuclease Activity:
A 50 µL reaction containing 15,000 cpm of a radiolabeled single-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C resulted in less than 0.1% release of TCA-soluble counts.
Double-Stranded Exonuclease Activity:
A 50 µL reaction containing 15,000 cpm of a radiolabeled double-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C resulted in less than 0.1% release of TCA-soluble counts.
RNAse Contamination:
A reaction containing 1 µg of an RNA ladder substrate and 10 µL of enzyme solution incubated for 2 hours at 37°C resulted in no detectable substrate degradation as measured by agarose gel electrophoresis.
E.coli 16S rDNA Contamination Test:
Replicate 5 µL samples of enzyme solution were 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. The acceptance criterion for the test is the threshold cycle count (Ct) produced by the average of 3 replicate no template control samples. Based on the correlation between the no template control Ct values, and standard curve data, the detection limit of this assay is <10 copies genome/sample.
Quality Control Analysis
Unit Characterization Assay:
Unit activity was measured using a 2-fold serial dilution method. Dilutions of enzyme were made in a glycerol (50%) containing T7 RNA Polymerase storage solution ([T7 RNA Polymerase]f = 64– 0.5 µg/µL) and added to 50 µL reactions containing 2 µg T7 promoter-containing plasmid DNA, 1X T7 RNA Polymerase Buffer, 2 µCi/mL α-32P-ATP and 400 µM NTPs. Reactions were incubated 10 minutes at 37°C, plunged on ice, and analyzed using the method of Sambrook and Russell (Molecular Cloning, v3, 2001, pp. A8.25-A8.26)
Protein Concentration (OD280) Measurement:
A 3.0 µL sample of enzyme was analyzed at OD280 using a Nanodrop ND-1000 spectrophotometer standardized using a 2.0 mg/ml BSA sample (Pierce Cat #23209) and blanked with product storage solution. The observed average measurement of 3 replicate samples was converted to mg/mL using an extinction coefficient of 138,830 and molecular weight of 98,855 Daltons. Acceptance for this assay is +/- 5% of reference sample.
SDS-Page (Physical Purity Assessment):
2.0 µL of enzyme solution was loaded on a denaturing 4-20% Tris-Glycine SDS-PAGE gel flanked by a broad-range MW marker and 2.0 µL of a 1:100 dilution of the sample. Following electrophoresis, the gel was stained and the samples compared to determine physical purity. The acceptance criteria for this test requires that the aggregate mass of contaminant bands in the concentrated sample do not exceed the mass of the protein of interest band in the dilute sample, confirming greater than 99% purity of the concentrated sample.
Recommended Storage Conditions:
-20°C
References:
- Chamberlin, M, et al. (1973) J. Biol. Chem. 248, 2235-2244, 2245-2250.
- Chamberlin, M. et al. (1982) in The Enzymes, 3rd edition, ed. P. D. Boyer (Academic Press, New York.) 15, 87-108.
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