SP6 RNA polymerase is a DNA dependent RNA polymerase that specifically recognizes the SP6 promoter sequence (5′-ATTTAGGTGACACTATAGAAGNG-3′). SP6 RNA polymerase can catalyze the incorporation of NTP of single stranded or double stranded DNA template downstream of SP6 promoter to synthesize RNA complementary to DNA template downstream of SP6 promoter. Linear plasmids containing SP6 promoters or PCR products containing SP6 promoters can be used as templates for RNA synthesis in vitro.
|5× RNA Polymerase reaction buffer||1mL||3mL||13mL|
This product is expressed by Escherichia coli, and the expression gene is phage SP6 RNA polymerase gene.
1. Preparation of RNA vaccines and drugs;
2. Synthesis of RNA for in vitro translation;
3. Preparation of radiolabeled RNA probes;
4. Synthesis of antisense RNA for experimental study of expression regulation;
5. Synthesis includes mRNA, siRNA, gRNA and other RNA precursors;
6. Synthesis of Capped mRNA with (Cap analog) as primer.
Store at -20 ℃ for 24 months.
50mM Tris-HCl(pH7.9), 100mM NaCl, 1mM EDTA, 20mM β- Mercaptoethanol, 0.1% Triton® X-100, 50% glycerin.
1× RNA polymerase reaction buffer composition
40mM Tris HCl (pH7.9), 10mM DTT, 6mM MgCl2, 2mM spermidine.
100μL system, one unit enzyme is defined as the amount of enzyme required to add 5 nmol of ATP into polynucleotides within 1 hour at 37 ℃.
Enzyme activity test conditions
50μL reaction system, it contains 1× RNA polymerase reaction buffer, 2mM NTP (0.5mM ATP, GTP, CTP, UTP respectively), 10mM DTT, 1μg DNA template with SP6 promoter.
SP6 RNA polymerase does not contain other nucleases, such as T7 or T3 RNA polymerase, DNase and RNA enzyme.
Recommended protocol for RNA synthesis in vitro
1. Template preparation: DNA template containing SP6 promoter and target gene (PCR amplification product, or linearized plasmid). If you use enzyme digestion to linearize the plasmid, it is better to use a restriction endonuclease with a flat end (such as EcoR V), or a sticky terminal enzyme with a protruding 5′ end (such as SapI or BspQI).
2. Precautions before experiment: Make sure that the entire experimental environment is free of RNA enzyme pollution. Use a nozzle, centrifuge tube, and ultrapure water that are free of RNA enzyme. Make sure that the entire reaction system is prepared without RNA enzyme pollution. Make sure that the experimental table, pipette gun, electrophoresis equipment, etc. are free of RNA enzyme pollution. Try to wear masks, hats, and gloves.
3. Reaction system configuration (recommended, system optimization recommended)
|5× RNA polymerase reaction buffer||10 μL|
|100 mM DTT (optional)||5 μL|
|NTP mix (ATP/CTP/GTP/UTP, 2.5mM each)||10 μL|
|Linearized DNA template||0.5~1 μ g|
|SP6 RNA Polymerase||1 μL|
|RNA enzyme inhibitor||50 units|
|DEPC treated water||to final volume 50 μL|
4. Incubate at 37 ℃ for 2h.
5. Use DNase I to remove template DNA contamination (optional): every 10μL system add 1U DNase I and incubate at 37 ℃ for 30min. DNase I can be inactivated by heating at 65 ℃ for 10min.
1. Simon Stammen, Franziska Schuller, Sylvia Dietrich, Martin Gamer, Rebekka Biedendieck, Dieter Jahnl (2010). Application of Escherichia coli phage K1E DNA-dependent RNA polymerase for in vitro RNA synthesis and in vivoprotein production in Bacillus megaterium. Appl Microbiol Biotechnol (2010) 88:529–539.
2. W.Tom Stump, Kathleen B. Hall(1993), SP6 RNA polymerase efficiently synthesizes RNA from short double-stranded DNA templates, Nucleic Acids Research, 5480-5484 Nucleic Acids Research, 1993, Vol. 21, No. 23.
3. Jiyun Yoo 1, Changwon Kang (2000), Bacteriophage SP6 RNA polymerase mutants with altered termination efficiency and elongation processivity, Genetic Analysis: Biomolecular Engineering 16 (2000) 191–197.
4. E D Jorgensen, R K Durbin, S S Risman, W T McAllister (1991), Specific contacts between the bacteriophage T3, T7, and SP6 RNA polymerases and their promoters, Volume 12, Issue 7, November 2019, Pages 908-931.
5. Hirokazu Kotaoi, Yukuo Ishizalci, Nobutsugu Hiraoka and AJrira Obayashi(1987),Nucleotide sequence and expression of the cloned gene of bacteriphage SP6 RNA polymerase, Volume 15 Number 6 1987.
|GMP-SP601||SP6 RNA Polymerase||GMP grade SP6 RNA Polymerase, 20U/uL|