Purified from an E.coli strain carrying a plasmid with the cloned gene encoding this enzyme. SP6 RNA Polymerase has a stringent specificity for its own promoter sequences and catalyzes the synthesis of RNA from ribonucleoside triphosphates in the presence of a DNA template.
Applications:Generation of strand-specific RNA sequences (see Protocol for Synthesis of Strand-specific RNA Sequences and Protocol for Synthesis of High Specific Activity Radiolabeled RNA Sequences) that may be used:Probes for hybridization (1) and genomic DNA sequencing (2), RNase protection assays (3), Antisense RNAs (4) Templates for in vitro RNA translation analysis (5) Substrates for studies of RNA splicing (6), RNA secondary structure and RNA-protein interactions (7).
Supplied with 20u/ul 0.25ml of 5X Transcription Buffer
Unit Definition:One unit of enzyme incorporates 1 nanomole of AMP into a polynucleotide fraction (adsorbed on DE-81) in 60 minutes at 37°C.
Activity Assay:40mM Tris-HCl (pH 8.0), 6mM MgCl2, 10mM DTT, 2mM spermidine, 0.5mM of each NTP, 0.6MBq/ml [3H]-ATP, 20ug/ml plasmid DNA containing the specific SP6 RNA Polymerase promoter sequence.
5X Transcription Buffer:200mM Tris-HCl (pH 7.9 at 25°C), 30mM MgCl2, 50mM DTT, 50mM NaCl and 10mM spermidine.
Quality Control:Endodeoxyribonuclease Assay:No detectable conversion of covalently closed circular DNA to nicked DNA was observed after incubation of 200 units of SP6 RNA Polymerase with 1ug of pBR322 DNA in 50ul of buffer (40mM Tris-HCl (pH 8.0), 6mM MgCl2, 10mM DTT) for 1 hour at 37°C.
Exodeoxyribonuclease Assay:0% of the total radioactivity was released into trichloroacetic acid-soluble fraction after incubation of 200 units of SP6 RNA Polymerase with 1ug of sonicated E.coli [3H]-DNA in 50ul of buffer (40mM Tris-HCl (pH 8.0), 6mM MgCl2, 10mM DTT) for 1 hour at 37°C.
Ribonuclease Assay:0% of the total radioactivity was released into trichloroacetic acid-soluble fraction after incubation of 200 units of SP6 RNA Polymerase with 1ug of E.coli [3H]-RNA in 50ul of buffer (40mM Tris-HCl (pH 8.0), 6mM MgCl2, 10mM DTT) for 1 hour at 37°C.
Functional Assay:SP6 RNA Polymerase was tested for use in the synthesis of strand-specific RNA sequences.
