Supplying a reaction lasting for at least two hours with additional chemicals every 20 minutes is quite uncomfortable, so we varied the initial concentration of Mg(OAc)2 in the reaction mix. From personal discussions with a leading scientist in the field of cell-free expression, we were adviced to adjust our mix to a concentration of 15-18 mM Mg2+. Therefore, we expressed pBESTlucTM with our DiaMIX in 50 µl reactions at concentrations between 12-16 mM to compare the expression via luciferase assay. The result is shown in figure 3 and reveals the optimal initial concentration of Mg(OAc)2 to be at 12 mM.