Templiphi amplification kit

For rolling circle amplification (RCA) of circular DNAs, EquiPhi29 polymerase (Thermo Fisher) was used with the denatured mix consisting of 4 μL of EquiPhi29 reaction buffer, 1 μL of extracted viral DNA, and 1 μL of each RCA primer listed in Table 1. The mixture was heated to 95 °C for 3 min and placed on ice for 5 min. A reaction mix was made consisting of 0.4 μL of 100 mM DTT (included in EquiPhi kit), 4 μL of 10 mM dNTPs (Thermo Fisher), 2 μL of EquiPhi29 polymerase, and 19.6 of μL water. The mix was vortexed, combined with the denatured mix, incubated at 45 °C for 3 h, and at 65 °C for 10 min to terminate the reaction.
For the TempliPhi procedure, the TempliPhi amplification kit (Cytiva) was used according to the manufacturer’s instructions. In short, 1 μL of extracted DNA and 5 μL of sample buffer were combined, denatured at 95 °C for 3 min, and placed on ice. A premix of 5 μL of reaction buffer and 0.2 μL of enzyme mix was combined. 5 μL of the premix was added to the sample mix and incubated at 30 °C for 18 h, followed by an inactivation step at 65 °C for 10 min.
Both reactions were performed in a thermocycler with a heated lid. Products of both reactions were stored at −20 °C for future use.