SP6 RNA Polymerase
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High concentration available Recombinant enzyme Thermal inactivation at 70°C in 10 min
SP6 RNA polymerase is DNA-dependent RNA polymerase catalyzes the 5'→3' synthesis of RNA on either single-stranded or double-stranded DNA under control of the SP6 promoter
Detailní popis
Thermo Scientific Bacteriophage SP6 RNA polymerase is a DNA-dependent RNA polymerase with strict specificity for its respective double-stranded promoters. It catalyzes the 5'→3' synthesis of RNA on either single-stranded DNA or double-stranded DNA downstream from its promoter and incorporates modified nucleotides.
Highlights
- Incorporates modified nucleotides (e.g., aminoallyl-, biotin-, fluorescein-, digoxigenin-labeled nucleotides)
Applications
- Synthesis of unlabeled and labeled RNA that can be used:
- For hybridization (see Reference 1), in vitro RNA translation (see Reference 2)
- As aRNA (see Reference 3), siRNA (see Reference 4), substrate in RNase protection assays (see Reference 5), template for genomic DNA sequencing (see Reference 6)
- In studies of RNA secondary structure and RNA-protein interactions (see Reference 7), RNA splicing (see Reference 8)
Note
Consensus promoter sequences:
T3 | AATTAACCCTCACTAAAGGGAGA |
---|---|
T7 | TAATACGACTCACTATAGGGAGA |
SP6 | ATTTAGGTGACACTATAGAAGNG |
The position in bold (+1) indicates the first nucleotide incorporated into RNA during transcription. Only bases at this position through +3 are critical for transcription, and they must be a G and a purine base, respectively (see Reference 9).
5× Transcription Buffer | 200 mM Tris-HCl (pH 7.9 at 25°C), 30 mM MgCl2, 50 mM DTT, 50 mM NaCl, 10 mM spermidine. |
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Definition of Activity Unit | One unit of the enzyme incorporates 1 nmol of AMP into a polynucleotide fraction (adsorbed on DE-81) in 60 minutes at 37°C. Enzyme activity is assayed in the following mixture: 40 mM Tris-HCl (pH 8.0), 6 mM MgCl2, 10 mM DTT, 2 mM spermidine, 0.5 mM of each NTP, 0.6 MBq/mL [3H]-ATP, 20 µg/mL plasmid DNA containing the appropriate promoter sequences. |
Hazardous | No |
Hazardous: | No |
Inactivation | Inactivated by heating at 70°C for 10 min or by addition of EDTA. |
Inhibition | Inhibitors: metal chelators, enzyme activity is reduced by 50% at NaCl or KCl concentration above 150 mM. Greater than 50% reduction in enzyme activity with ammonium sulphate. |
Molecular Weight | 99 kDa monomer |
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Source | E. coli cells with a cloned gene encoding SP6 RNA polymerase. |
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Storage Buffer | Polymerase is supplied in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM DTT, 0.1 mg/mL BSA, 0.03% (v/v) ELUGENT Detergent, 50% (v/v) glycerol. |
Storage Condition | -20 C |
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