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.