FAQ: What if I want to use TtAgo with Mn²⁺ [manganese(II)] instead of Mg²⁺ [magnesium(II)] as provided in ThermoPol^® Reaction Buffer?

TtAgo can utilize either Mg²⁺ or Mn²⁺ in its active site. The typical reaction conditions in the provided NEB ThermoPol^® Reaction Buffer are amenable to spiking Mn²⁺ into the reaction in the presence of 2mM Mg²⁺ with similar effect as with Mn²⁺ alone. However, if no Mg²⁺ is desired in the reaction, a custom buffer can be prepared. We recommend using 20 mM Tris-HCl pH 8.8 buffer with low salt (approximately 10 mM NaCl or KCl) and adding Mn²⁺ to the desired concentration (typically in the range of 0.1–2mM). A similar trend of slightly higher overall performance accompanied by off-target activity is observed when using TtAgo with Mn²⁺ as with Mg²⁺, however Mn²⁺ has lower range (0.1 to 2mM) of working concentrations compared to Mg²⁺ (0.5 to 5mM). For more information, please refer to GUIDElines for optimization of Tth Argonaute (TtAgo) reactions.