T7 (NEB # M0251) and SP6 (NEB # M0207) RNA polymerases are DNA dependent RNA polymerases that produce DNA templated RNA transcripts. T7 and SP6 exhibit high specificity for their respective promoters. Both T7 and SP6 can be used for the in vitro synthesis of RNA for a wide variety of applications, including transfection, translation, structural studies and radioactive and non-isotopic probe generation. E. coli Poly(A) Polymerase (NEB # M0276) and Poly(U) Polymerase (NEB # M0337) generate untemplated homoribopolymeric tails on the 3´-ends of RNA. Both E. coli Poly(A) Polymerase and Poly(U) Polymerase can be used for RNA tailing for reverse transcription or labeling.
In vitro RNA synthesis requires a DNA template, RNA polymerase, NTPs and other factors. High-yield robust reactions require optimization of each reaction component. NEB offers five in vitro RNA synthesis kits, all of which have been rigorously formulated to provide reproducible high yields of quality RNA. Additionally, NEB offers a Vaccinia capping system (NEB # M2080) and a number of RNA cap analogs.
- A Typical DNase I Reaction Protocol (M0303)
- Capped RNA Synthesis (E2040)
- Capping Protocol (M2080)
- DNA Template Preparation (E2040)
- Evaluation of Reaction Products (E2040)
- High Specific Activity Radiolabeled RNA Probe Synthesis (E2040)
- A Typical Tailing Reaction (M0337)
- Purification of Synthesized RNA (E2040)
- RNA Synthesis with Modified Nucleotides (E2040)
- SP6 In Vitro Transcription Reaction Protocol (M0207)
- Standard RNA Synthesis (E2040)
- 2´-O-Methylation of Capped RNA (M0366)
- One-Step Capping and 2´-O-Methylation (M0366)
- Labeling Protocol (M2080)
- Capped RNA Synthesis (E2050)
- Evaluation of Reaction Products (E2050)
- Purification of Synthesized RNA (E2050)
- RNA Synthesis with Modified Nucleotides (E2050)
- Standard RNA Synthesis (E2050)
- In vitro digestion of DNA with Cas9 Nuclease, S. pyogenes (M0386)
- Poly(A) Tailing of RNA using E. coli Poly(A) Polymerase (NEB# M0276)
- Protocol for Standard RNA Synthesis
- Evaluation of Reaction Products (E2060)
- mRNA Purification (E2060)
- mRNA Purification (E2065)
- mRNA Synthesis with Modified Nucleotides (E2060)
- mRNA Synthesis with Modified Nucleotides (E2065)
- Standard mRNA Synthesis (E2060)
- Standard mRNA Synthesis (E2065)
- Evaluation of Reaction Products (E2065)
- Using recombinant Cas9 nuclease to assess locus modification in genome editing experiments (#M0386)
- sgRNA Synthesis Using the HiScribe™ Quick T7 High Yield RNA Synthesis Kit (NEB #E2050)
- EnGen® sgRNA Synthesis Kit, S. pyogenes Protocol (E3322)
- Transfection of Cas9 RNP (ribonucleoprotein) into adherent cells using the Lipofectamine® RNAiMAX
- Purification of Synthesized RNA (E2070)
- RNA Synthesis Protocols (E2070)
- Avoiding Ribonuclease Contamination
- Potapov, V., Fu, X., Dai, N., Correa, I.R., Jr., Tanner, N.A., Ong, J.L 2018. Base modificatons affecting RNA polymerase and reverse transcriptase fidelity Nucleic Acids Research. 46(11): 5753-5763, PubMedID: 29750267, DOI:
- Eyler, D.E., Franco, M.K., Batool, Z., Wu, M.Z., Dubuke, M.L., Dobosz-Bartoszek, M., Jones, J.D., Polikanov, Y.S., Roy, B., Koutmou, K.S 2019. Pseudouridinylatio of mRNA coding sequences alters translation Proc Natl Acad Sci USA. 116(46), PubMedID: 31672910, DOI: 10.1073/pnas.1821754116
- Wu, M.Z., Asahara, H., Tzertzinis, G., Roy, B. 2020. Synthesis of low immunogenicity RNA with high-temperature in vitro transcription . , PubMedID: 31900329, DOI:
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This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene.