New England Biolabs, M0207S, SP6 RNA Polymerase

SP6 RNA Polymerase is used for Related Categories cDNA Synthesis & Reverse Transcriptases,, RNA Synthesis In vitro Transcription (IVT),, Nucleotide Solutions for RNA Applications PCR Specification Materials Required but not Supplied RNase Inhibitor, Murine (NEB #M0314) (optional) Ribonucleotide Solution Mix (NEB #N0466) Fresh DTT (optional) Nuclease-free Water (NEB #B1500) Unit Definition One unit is defined as the amount of enzyme required to incorporate 1 nmol ATP into an acid-insoluble material in 1 hour at 37°C. Reaction Conditions 1X RNAPol Reaction Buffer Supplement with 0.5 mM ATP, 0.5 mM GTP, 0.5 mM UTP and 0.5 mM CTP 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 DNA containing the SP6 promoter in 50 μl. FAQ Q: Does the transcription reaction with SP6 RNA Polymerase require a primer? A: No, SP6 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 SP6 RNA Polymerase leave an extra base at the end of a transcript? A: Yes, SP6 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 SP6 RNA Polymerase work on single stranded substrate? A: No, the promoter sequence region must be double stranded. Q: Will SP6 RNA Polymerase work on uncut plasmid DNA? A: Yes, but SP6 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 SP6 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: Can I use SP6 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: 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. Q: What are the main causes of reaction failure using SP6 RNA Polymerase? A: SP6 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.