We recommend two possible variant approaches to making master mixes: One for when trying different oligo combinations, and one for testing different templates once the oligonucleotides are chosen. Changing the order of addition for these reactions can lead to sub-optimal RPA reactions. If using the TwistAmp® Liquid exo kit, it is best to keep the 50x Exo reagent separate from oligos/template until they have been coated in ssDNA binding proteins (present in the Core Reaction Mix), or they may be digested. MgAc should always be added last as it starts the reactions instantly and will begin the consumption of ATP even if no template has been added. Oligonucleotides should all be introduced to the Core Reaction Mix simultaneously to prevent preferential binding of the oligonucleotide introduced first.