New England Biolabs, M2080S, Vaccinia Capping System

GMP-grade reagent also available. Related Categories RNA Capping Specification Unit Definition One unit of Vaccinia Capping Enzyme is defined as the amount of enzyme required to incorporate 10 pmol of (α 32 P) GTP into an 80 nt transcript in 1 hour at 37°C. Reaction Conditions 1X Capping Buffer Supplement with 0.5 mM GTP and 0.1 mM S-adenosylmethionine (SAM) Incubate at 37°C 1X Capping Buffer 50 mM Tris-HCl 5 mM KCl 1 mM MgCl2 1 mM DTT (pH 8 @ 25°C) Storage Buffer 20 mM Tris-HCl 100 mM NaCl 1 mM DTT 0.1 mM EDTA 50% Glycerol 0.1% (w/v) Triton® X-100 pH 8 @ 25°C FAQ Q: What is Cap-0 and Cap-1? A: Cap-0 is a N7-methyl guanosine connected to the 5′ nucleotide through a 5′ to 5′ triphosphate linkage, typically refers to as m7G cap or m7Gppp- in the literature. In the cell, the Cap-0 structure is essential for efficient translation of the mRNA that carries the cap. An additional methylation on the 2′O position of the initiating nucleotide generates Cap-1, or refers to as m7GpppNm-, where Nm denotes any nucleotide with a 2′O methylation. Cap-1 has been shown to be important in evading the cellular innate immune response in vivo. Q: Can Vaccinia Capping Enzyme (NEB #M2080) use cap analogs in capping reaction? A: No. Vaccinia Capping Enzyme uses GTP and SAM to generate a cap structure on 5′ triphosphate RNA, typically a synthetic transcript generated by in vitro transcription. On the other hand, cap analogs, such as GpppG (NEB #S1407), m7GpppG (NEB #S1404), m7GpppA (NEB #S1405S) and anti-reverse cap analog (ARCA; NEB #S1411) are used during in vitro transcription, where the cap analog is directly incorporated into the transcript as the first nucleotide at the 5′ end.