{"product_id":"neb-e2070s","title":"New England Biolabs, E2070S, HiScribe® SP6 RNA Synthesis Kit","description":"\u003cb\u003eRelated Categories\u003c\/b\u003e RNA Capping,, RNA Synthesis In vitro Transcription (IVT),, Nucleotide Solutions for RNA  \u003cb\u003eSpecification\u003c\/b\u003e \u003cb\u003eMaterials Required but not Supplied\u003c\/b\u003e Nuclease-free Water (NEB #B1500)  \u003cb\u003eFAQ\u003c\/b\u003e Q: What is the promoter sequence for SP6 RNA Polymerase? A: The SP6 promoter sequence is 5´ ATTTAGGTGACACTATAG 3´. SP6 RNA Polymerase starts transcription at the underlined G in the double-stranded promoter sequence. The polymerase then transcribes using the opposite strand as a template in the 5´ to 3´ direction. The first base in the transcript will be a G. Q: Does transcription with SP6 RNA Polymerase require a primer? A: No. Unlike DNA polymerases, no primers are needed for the activity of SP6 RNA Polymerase. The SP6 promoter region must be double-stranded in order for the polymerase to recognize the promoter sequence with transcription starting at the final G. The polymerase then transcribes using the opposite strand as the template from 5´ to 3´. Linearized plasmids, PCR products or synthetic DNA oligonucleotides annealed to a short, SP6 promoter-containing oligonucleotide can all serve as templates for in vitro transcription with SP6 RNA Polymerase. Q: Does SP6 RNA Polymerase leave an extra base at the end of the transcript? A: Yes, SP6 RNA Polymerase can leave one or a few extra bases at the 3´ end of the transcript resulting in a pool of transcripts with heterogeneous 3´ ends. Q: Will SP6 RNA Polymerase initiate transcription from a single-stranded DNA template? A: No. The promoter region must be double-stranded for the polymerase to recognize it and initiate transcription. The polymerase then transcribes using the opposite strand as a template in the 5´ to 3´ direction. Q: Will SP6 RNA Polymerase work on an uncut plasmid? A: Yes, though SP6 is an extremely processive enzyme and will continue to transcribe around a circular template multiple times without disassociating. The plasmid can be linearized with restriction enzymes that leave a blunt or 5´ overhang downstream of the DNA of interest, resulting in run-off transcription. Q: Can aberrant RNA be produced using SP6 RNA polymerase? A: Yes. There have been reports that aberrant RNA is produced when linearizing a plasmid using restriction enzymes that leave 3´ overhangs. To avoid this, the plasmid should be linearized using restriction enzymes that leave a blunt or 5´ overhang just downstream of the DNA of interest. Q: Can kit components from the HiScribe SP6 RNA Synthesis Kit be substituted with SP6 RNA Polymerase and Reaction Buffer (NEB #M0207)? A: No. The kit components have been specially formulated and should be used with provided protocols for optimal results. The formulations are different from the enzyme and buffer included in NEB #M0207 and cannot be substituted. Q: What is the sequence of the SP6 Control Template? A: The sequence of the SP6 Control Template, (pCLuc-SP6) which contains the Cypridina luciferase gene under control of the SP6 promoter, can be found in the DNA Sequences and Maps Tool on neb.com. Q: Can I use the Monarch Spin RNA Cleanup Kits to cleanup my in vitro transcription (IVT) reaction? A: Yes, the Monarch Spin RNA Cleanup Kit (50 µg) (NEB #T2040) is suitable for cleaning up in vitro transcription reactions where the yield is not expected to exceed 50 µg. If the RNA yield of an in vitro transcription is expected to exceed 50 µg, we recommend the reaction be split among multiple Monarch Spin RNA Cleanup Columns (50 µg) or cleaned up using the Monarch Spin RNA Cleanup Kit (500 µg) (NEB #T2050), due to the larger RNA binding capacity. Q: Are modified nucleotides included in the kit? A: Modified nucleotides are not included in the kit but can be purchased separately. NEB can provide N1-Methyl-Pseudouridine-5’-Triphosphate (NEB #N0431), 5-Methyl-Cytidine-5’-Triphosphate (NEB #N0432), Pseudouridine-5’-Triphosphate (NEB #N0433) and 5-Methoxy-Uridine-5’-Triphosphate (NEB #N0434). The kit manual includes a detailed protocol for using modified nucleotides in the transcription reaction. Q: Do I need to add DTT to the reaction? A: Addition of DTT (5mM final) to the reaction is optional but recommended.The RNA polymerase in the kit is sensitive to oxidation and could result in lower RNA yield over time due to repeated handling etc. Adding DTT to the reaction may help restore the kit performance in such cases. Adding DTT will not compromise the reaction in any situation. ","brand":"New England Biolabs","offers":[{"title":"Default Title","offer_id":46835478921385,"sku":"E2070S","price":0.99,"currency_code":"USD","in_stock":true}],"url":"https:\/\/iright.com\/products\/neb-e2070s","provider":"Iright","version":"1.0","type":"link"}