SplintR Ligase has been developed for the life sciences community, with a strong appreciation for its utility as a detection reagent. Some empirical optimization is likely necessary for individual applications. We employed a very sensitive assay, in which the substrate is present in nanomolar amounts. As such, the unit definition is targeted for applications where single nanogram amounts of substrate are routinely employed. The enzyme is supplied as a 10.5 uM solution. We suggest maintaining the enzyme below 1 uM in the reaction with a suggested range of 100 nM to 1 uM. For many applications, starting with a 2-fold excess of enzyme over ligatable ends is ideal. Additonally, if the reaction is not proceeding as efficiently as desired, we strongly recommend extending the incubation time rather than increasing the amount of enzyme in the reaction.