RNA Synthesis
RNA Polymerases
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.
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Protocols for RNA Synthesis
- RNA Synthesis with Modified Nucleotides (E2050)
- Standard RNA Synthesis (E2050)
- Purification of Synthesized RNA (E2050)
- Capped RNA Synthesis (E2050)
- Evaluation of Reaction Products (E2050)
- sgRNA Synthesis Using the HiScribe™ Quick T7 High Yield RNA Synthesis Kit (NEB #E2050)
- mRNA Synthesis with Modified Nucleotides (E2065)
- Standard mRNA Synthesis (E2060)
- Standard mRNA Synthesis (E2065)
- mRNA Synthesis with Modified Nucleotides (E2060)
- Evaluation of Reaction Products (E2060)
- mRNA Purification (E2060)
- mRNA Purification (E2065)
- Evaluation of Reaction Products (E2065)
- Protocol for Standard RNA Synthesis
- RNA Synthesis Protocols (E2070)
- Purification of Synthesized RNA (E2070)
- Standard RNA Synthesis (E2040)
- Purification of Synthesized RNA (E2040)
- RNA Synthesis with Modified Nucleotides (E2040)
- Capped RNA Synthesis (E2040)
- High Specific Activity Radiolabeled RNA Probe Synthesis (E2040)
- Evaluation of Reaction Products (E2040)
- DNA Template Preparation (E2040)
- SP6 In Vitro Transcription Reaction Protocol (M0207)
- Capping Protocol (M2080)
- A Typical DNase I Reaction Protocol (M0303)
- Labeling Protocol (M2080)
- 2´-O-Methylation of Capped RNA (M0366)
- One-Step Capping and 2´-O-Methylation (M0366)
- EnGen® sgRNA Synthesis Kit, S. pyogenes Protocol (E3322)
- In vitro digestion of DNA with Cas9 Nuclease, S. pyogenes (M0386)
- Transfection of Cas9 RNP (ribonucleoprotein) into adherent cells using the Lipofectamine® RNAiMAX
- Electroporation of Cas9 RNP (ribonucleoprotein) into adherent cells using the Neon® Electroporator
- A Typical Tailing Reaction (M0337)
- Using recombinant Cas9 nuclease to assess locus modification in genome editing experiments (#M0386)
- Poly(A) Tailing of RNA using E. coli Poly(A) Polymerase (NEB# M0276)
- Avoiding Ribonuclease Contamination
Other Tools & Resources
Usage Guidelines
RNA Reagents Overview
Recommended HiScribe RNA Synthesis Kits by Application
RNA Polymerase Selection Chart
RNA Ligase Selection Chart
Cap Analog Selection Chart
Videos
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In Vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection
This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene.
