New England Biolabs, M0658S, Hi-T7® RNA Polymerase

Hi-T7 RNA Polymerase is designed for Related Categories RNA Synthesis In vitro Transcription (IVT),, Nucleotide Solutions for RNA Specification Materials Required but not Supplied RNase Inhibitor, Murine (NEB #M0314) (optional) Ribonucleotide Solution Mix (NEB #N0466) Nuclease-free Water (NEB #B1500) Unit Definition One unit is defined as the amount of enzyme required to incorporate 1 nmol of ATP into acid-insoluble material in 1 hour at 50°C. Reaction Conditions 1X Hi-T7 RNA Polymerase Reaction Buffer, supplemented with 0.5 mM each of ATP, UTP, GTP, CTP, and DNA template containing the T7 RNA Polymerase promoter. Incubate at 37-56°C for 1 hour. Optimal incubation temperature is 50-52°C. Reaction Conditions 1X Hi-T7® RNA Polymerase Reaction Buffer Supplement with 0.5 mM ATP, 0.5 mM UTP , 0.5 mM GTP and 0.5 mM CTP Incubate at 50°C 1X Hi-T7® RNA Polymerase Reaction Buffer 40 mM Tris-HCl 4 mM MgCl2 50 mM NaCl 2 mM spermidine 1 mM DTT (pH 7.5 @ 25°C) Storage Buffer 50 mM Tris-HCl 100 mM NaCl 1 mM EDTA 1 mM DTT 0.1% (w/v) Triton® X-100 50% Glycerol pH 7.9 @ 25°C Unit Assay Conditions 1X Hi-T7 RNA Polymerase Reaction Buffer, supplemented with 0.5 mM each ATP, UTP, GTP, CTP and 1 µg T7 phage DNA in 50 μl reaction volume. FAQ Q: Can I use a different reaction buffer for in vitro transcription with Hi-T7 RNA Polymerase? A: No. The Hi-T7 RNA Polymerase Reaction Buffer has been formulated to work specifically with Hi-T7 RNA Polymerase. Q: Does Hi-T7 RNA Polymerase recognize the same promoter sequence as the wild-type T7 RNA Polymerase from bacteriophage? A: Yes. Hi-T7 and T7 RNA Polymerases recognize the same promoter: 5′ TAATACGACTCACTATAG 3′ with transcription starting at the G. The first base in the transcript will be a G. Q: At what temperature is Hi-T7 RNA Polymerase active? A: Hi-T7 RNA Polymerase is active between 37°C and 56°C. When performing transcription reactions at temperatures higher than the suggested 50°C, it is recommended to limit the reaction time to 30 minutes or less as incubation of RNAs at high temperature for prolonged periods in the presence of MgCl2 (a component of the Hi-T7 RNA Polymerase Reaction Buffer) may lead to RNA degradation. Q: What are the main causes of reaction failure when using Hi-T7 RNA Polymerase? A: The purity of the DNA template is very important and should be prepared by purification methods such as spin column or phenol:chloroform extraction. In addition, to avoid RNase contamination we recommend wearing gloves and using filter tips and RNase-free tubes. It is important to mix the reaction components well after thawing and to set the reaction up at room temperature in the order listed in the protocol for optimal yield. Q: What templates can be used for in vitro transcription with Hi-T7 RNA Polymerase? A: Double-stranded DNA that can be used as templates with Hi-T7 RNA Polymerase include linearized plasmids, PCR products, or annealed complementary oligos that contain the T7 promoter sequence. Q: Can Hi-T7 RNA Polymerase transcribe off of an uncut plasmid? A: Yes. Hi-T7 RNA Polymerase will transcribe around a circular templates multiple times without disassociating. To transcribe an RNA of a specific length, the plasmid can be linearized with a restriction enzyme that leaves a blunt or 5′ overhang downstream of the DNA of interest.