{"product_id":"neb-m0378s","title":"New England Biolabs, M0378S, T3 RNA Polymerase","description":"Bacteriophage T3 RNA Polymerase is a DNA-dependent RNA polymerase that is highly specific for the T3 phage promoter.  \u003cb\u003eRelated Categories\u003c\/b\u003e cDNA Synthesis \u0026amp; Reverse Transcriptases,, RNA Synthesis In vitro Transcription (IVT),, Nucleotide Solutions for RNA  \u003cb\u003eApplications\u003c\/b\u003e PCR  \u003cb\u003eSpecification\u003c\/b\u003e \u003cb\u003eMaterials Required but not Supplied\u003c\/b\u003e RNase Inhibitor, Murine (NEB #M0314) (optional) Ribonucleotide Solution Mix (NEB #N0466) Fresh DTT (optional) Nuclease-free Water (NEB #B1500)  \u003cb\u003eUnit Definition\u003c\/b\u003e 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 Condition 1X RNAPol Reaction Buffer, supplemented with 0.5 mM each ATP, UTP, GTP, CTP and DNA template containing the T3 RNA Polymerase promoter. Incubate at 37°C.  \u003cb\u003eReaction Conditions\u003c\/b\u003e 1X RNAPol Reaction Buffer 1X RNAPol Reaction Buffer 40 mM Tris-HCl 6 mM MgCl2 1 mM DTT 2 mM spermidine (pH 7.9 @ 25°C)  \u003cb\u003eStorage Buffer\u003c\/b\u003e 100 mM NaCl 50 mM Tris-HCl 1 mM EDTA 20 mM 2-mercaptoethanol 0.1% Triton® X-100 50% (w\/v) Glycerol pH 7.9 @ 25°C  \u003cb\u003eUnit Assay Conditions\u003c\/b\u003e 1X RNAPol Reaction Buffer, supplemented with 0.5 mM each ATP, UTP, GTP, CTP and 2 μg T3 DNA in 50 μl.  \u003cb\u003eFAQ\u003c\/b\u003e Q: What is the promoter sequence of T3 RNA Polymerase? A: T3 Promoter 5′ AATTAACCCTCACTAAAG 3′ T3 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: Does the transcription reaction with T3 RNA Polymerase require a primer? A: No, T3 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 T3 RNA Polymerase leave an extra base at the end of a transcript? A: Yes, T3 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 T3 RNA Polymerase work on uncut plasmid DNA? A: Yes, but T3 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 T3 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 T3 RNA Polymerase to make high specific activity radiolabeled probes? A: Yes, 3RNA Polymerase, SP6 RNA Polymerase and T7 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 T3 RNA Polymerase? A: T3 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 #M0314 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. ","brand":"New England Biolabs","offers":[{"title":"Default Title","offer_id":46835484852393,"sku":"M0378S","price":0.99,"currency_code":"USD","in_stock":true}],"url":"https:\/\/iright.com\/products\/neb-m0378s","provider":"Iright","version":"1.0","type":"link"}