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Protocols for phi6 RNA Polymerase (RdRP) FAQs for Polymerases Technical Reference for Polymerases Enzyme Finder NEBcutter NEBuffer Chart Double Digest Finder Isoschizomers DNA Sequences and Maps REBASE HiScribe RNAi Transcription Kit Ribonucleotide Solution Mix

Home > Products > Polymerases > Reverse Transcriptases and RNA Polymerases > phi6 RNA Polymerase (RdRP) phi6 RNA Polymerase (RdRP) Catalog # Size Concentration Price Qty   M0255L 300 units 1,000 units/ml $360.00 M0255S 60 units 1,000 units/ml $90.00 Prices are in US dollars and valid only for US orders. Download: MSDS PDF Synthesis of dsRNA for RNAi experiments Synthesis of dsRNA from the 3' end of a ssRNA template Highly processive primer independent RNA synthesis Uses a short 9 nt initiator sequence Description: The phi6 RNA polymerase is a modified version of the P2 protein of bacteriophage phi6. It is an RNA-dependent RNA polymerase (RdRp) which synthesizes a full length complementary strand of an RNA initiating from the 3´ end resulting in dsRNA. The polymerase does not require any oligonucleotide primer for initiation. phi6 RdRp is a highly processive enzyme that can use either single or double stranded RNA template. The template strand of RNA requires the sequence 5´ NNUUUUUUUUCC 3´ for optimal initiation (1-3). Source: An E. coli strain that carries a modified P2 gene from bacteriophage phi6. Applications: Synthesis of dsRNA in vitro Synthesis of dsRNA for RNAi Reagents Supplied: phi6 RdRp Reaction Buffer (10X) MnCl2 (50 mM) Reaction & Storage Conditions Reaction Conditions: 1X phi6 RdRp Reaction Buffer Supplemented with 1.5 mM MnCl2 Incubate at 32°C. 1X phi6 RdRp Reaction Buffer: 50 mM Tris-HCl 50 mM Ammonium Acetate pH 8.9 @ 25°C Unit Definition: One unit is defined as the amount of enzyme that will incorporate 1 nmol of dUTP into an acid-insoluble material in 20 minutes at 32°C.  Unit Assay Conditions:  1 X phi6 RdRp Reaction Buffer, 1.5 mM MnCl2, 10% DMSO, 1 mM dUTP, 1 µg poly (rA) and 1 µCi 3H-UTP in 30 µl. Concentration: 1,000 units/ml Storage Conditions: 50 mM Tris-HCl 100 mM NaCl 0.1 mM EDTA 50% Glycerol 0.1% Triton X-100 pH 8.0 @ 25°C Storage Temperature: -20°C Notes General notes: Reaction Conditions: 1X phi6 RdRp Reaction Buffer, supplemented with 1.5 mM MnCl2, 0.1-0.2 mM each ATP, UTP, CTP and 0.3-0.6 mM GTP. Quality Control for Current Lot Quality control values for a specific lot can be found on the datacard which accompanies each product. Quality Assurance Statement: Phi6 RNA Polymerase (RdRp) has been purified free of exonucleases, endonucleases and RNases by Finnzymes Oy (Finland) (patent pending), a strategic partner of New England Biolabs. Quality controls are performed at Finnzymes Oy and confirmed at New England Biolabs. Endonuclease Activity: Incubation of a 50 μl reaction containing 1 units of phi6 RNA Polymerase (RdRP) with 1 μg of ΦX174 RF I DNA for 4 hours at 37ºC resulted in < 5% conversion to RFII as determined by agarose gel electrophoresis. RNase Assay: Incubation of a 50 μl reaction containing 1 units of phi6 RNA Polymerase (RdRP) with 1 μg of MS2 RNA for 1 hour at 37ºC resulted in no detectable degradation of the RNA as determined by agarose gel electrophoresis. Exonuclease Assay: Incubation of 1 unit of Phi 6 RNA Polymerase (RdRP) with 1 µg sonicated [3H] ssDNA (3 x 105 cpm/µg) for 4 hours at 37°C in 50 µl reaction buffer released < 0.5% acid soluble counts. References Makeyev, E.V. and Bamford, D.H. (2000) EMBO J., 19, 124-133. Makeyev, E.V. and Bamford, D.H. (2000) EMBO J., 19, 6275-6284. Laurila, M.R.L. et al. (2002) J. Biol. Chem., 277, 19117-17124. Companion Products HiScribe RNAi Transcription Kit Ribonucleotide Solution Mix