New England Biolabs, M0460T, T7 RNA Polymerase (High Concentration)

transcription using T7 phage promoter Related Categories RNA Synthesis In vitro Transcription (IVT),, Nucleotide Solutions for RNA Applications Transcription-Mediated and NASBA Amplification Specification Unit Definition One unit is defined as the amount of enzyme that will incorporate 1 nmol ATP into acid-insoluble material in a total reaction volume of 50 μl in 1 hour at 37°C. Reaction Conditions 1X RNAPol Reaction Buffer Incubate at 37°C 1X RNAPol Reaction Buffer 40 mM Tris-HCl 6 mM MgCl2 1 mM DTT 2 mM spermidine (pH 7.9 @ 25°C) Storage Buffer 50 mM Tris-HCl 100 mM NaCl 20 mM β-ME 1 mM EDTA 50% Glycerol 0.1% (w/v) Triton® X-100 pH 7.9 @ 25°C Unit Assay Conditions 1X RNAPol Reaction Buffer, supplemented with 0.5 mM each ATP, UTP, GTP, CTP, and 1 µg T7 DNA in 50 μl. FAQ Q: What is the promoter sequence of T7 RNA Polymerase? A: T7 Promoter 5′ TAATACGACTCACTATAG 3′ T7 RNA polymerase starts transcription at the underlined G in the promoter sequence. The polymerase then transcribes using the opposite strand as a template from 5’->3’. The first base in the transcript will be a G. Q: Is it possible to start transcription with an A? A: T7 RNA Polymerase can start transcription with an A under the class II T7 promoter. Similar to the regular T7 promoter, G is preferred at 2nd and 3rd position. T7 Promoter class II 5'- TAATACGACTCACTATTA 3' Q: Does the transcription reaction with T7 RNA Polymerase require a primer? A: No, T7 RNA Polymerase recognizes its specific promoter sequence and starts transcription at the final G. The polymerase then transcribes using the opposite strand as a template from 5’->3’. Q: Does T7 RNA Polymerase leave an extra base at the end of a transcript? A: Yes, T7 RNA Polymerase can add one or a few extra bases at the end of a transcript producing a pool of transcripts with heterogeneous 3’ ends. Q: Will T7 RNA Polymerase work on single stranded substrate? A: No, the promoter sequence region must be double stranded. Q: Will T7 RNA Polymerase work on uncut plasmid DNA? A: Yes, but T7 RNA Polymerase is an extremely processive enzyme and will continue to transcribe around a circular template multiple times without disassociating. The plasmid can be linearized with a restriction enzyme that leaves a blunt or 5' overhang downstream of the DNA of interest. Q: Can aberrant RNA be produced when using T7 RNA Polymerase? A: Aberrant RNA has been noted when the restriction enzyme used to linearize the plasmid downstream of the DNA of interest produces a 3' overhang. To avoid this, the plasmid should be cut with a restriction enzyme that leaves a blunt or 5' overhang downstream of the DNA of interest. Q: How can the yield of RNA be maximized when using T7 RNA Polymerase? A: Higher yields of RNA may be obtained by raising NTP concentrations (up to 4 mM each). Mg2+ concentration should be raised to 4 mM above the total NTP concentration. Additionally, inorganic pyrophosphatase can be added to a final concentration of 4 units/ml to lower feedback inhibition. Q: Can I use T7 RNA Polymerase to make high specific activity radiolabeled probes? A: Yes, T7 RNA Polymerase, SP6 RNA Polymerase and T3 RNA Polymerase and the HiScribe T7 Kit (NEB #E2040) can be used to make RNA probes. Higher detection sensitivity of nucleic acid can be obtained using an RNA probe due to the greater stability of RNA/DNA hybrids than DNA/DNA hybrids. Q: What are the main causes of reaction failure using T7 RNA Polymerase? A: T7 RNA Polymerase is extremely sensitive to salt inhibition. Monovalent salt concentration should not exceed 20mM. DNA template should be prepared free of salt. RNase contamination degraded the RNA product. We recommend using RNase inhibitor (NEB #0314 or #M0307) in reaction at 1 unit/ul. Use ultra-pure water and make sure the DNA template has been phenol/chloroform extracted. Q: Why is the specific activity of the probe low? A: The concentration of the radioactive nucleotide should be made limiting (6 μM). In other words, if using labeled ATP, add 500 μM of UTP, CTP, and GTP but only 6 μM ATP in the transcription so ATP is incorporated at higher percentage resulting very hot RNA probe.