New England Biolabs, M0458S, RtcB Ligase

Related Categories RNA Ligation Applications RNA Cloning Specification Reaction Conditions 1X RtcB Reaction Buffer Supplement with 0.1 mM GTP and 1 mM MnCl2 Incubate at 37°C 1X RtcB Reaction Buffer 50 mM Tris-HCl 75 mM KCl 3 mM MgCl2 10 mM DTT (pH 8.3 @ 25°C) Storage Buffer 50 mM NaCl 10 mM Tris-HCl 0.1 mM EDTA 1 mM DTT 50% Glycerol pH 7.4 @ 25°C FAQ Q: Does secondary structure affect ligation by RtcB Ligase? A: Yes. RtcB Ligase requires the 3’ end of the donor RNA to be single stranded in order to ligate efficiently. Double stranded substrates or a splinting oligo will block ligation. Q: Can RtcB Ligase ligate DNA? A: Yes. Published studies and internal testing has shown that RtcB Ligase is capable of ligating DNA donors to DNA acceptors or RNA donors to DNA acceptors, albeit with reduced efficiency. The same structural constraints apply to DNA substrates as for RNA substrates. Q: What is the optimum reaction temperature for RtcB Ligase? A: The optimum reaction temperature for RtcB ligase is 37°C. Q: Can RtcB Ligase be used in other NEBuffers? A: While RtcB Ligase is optimally active in the supplied buffer, it is also active in NEBuffer 3 and 1 at 30% and 10% activity, respectively. Use of these buffers requires the addition of 0.1 mM GTP and 1 mM MnCl2. Q: How can I increase ligation efficiency? A: Extending the ligation time or addition of more enzyme will increase ligation efficiency. Q: Can magnesium, or another divalent cation, be substituted for manganese? A: No. RtcB Ligase activity specifically requires manganese and is not replaceable by any other divalent cation. Q: Does PEG improve ligation efficiency? A: Under recommended conditions PEG8000 provides no benefit when equimolar amounts of ligase and substrate are used. When substrate is in excess of enzyme, addition of 15% PEG8000 provides maximum improvement in ligation efficiency. Q: Can I purchase large amounts of an existing Enzyme for Innovation? A: Yes, if you have use for large amounts of an Enzyme for Innovation, please contact NEB at EnzymesForInnovation@neb.com. Please note that there may be limited availability and thus extended lead times required for large orders. Additional QC testing may also be necessary to meet your specific application and needs. Q: What are Enzymes for Innovation? A: Enzymes for Innovation (EFI) is a project initiated by NEB to provide unique enzymes to the scientific community in the hopes of enabling the discovery of new and innovative applications. These enzymes have interesting properties and unique specificities that are not commercially available elsewhere at the quality that you would expect from NEB. Enzymes for Innovation graduates are enzymes that have transitioned from having unknown or exploratory applications to becoming the cornerstone of a defined application. If you have an idea for an “Enzyme for Innovation” with a suggested application that may be useful, please email us at EnzymesForInnovation@neb.com. For more information, please view video.