When performing an assay with UNG, a first step at 50°C during 2min and a second step at 95°C during 10min has to be added before performing a normal qPCR:
50 °C during 2min
95 °C during 10min
94 °C during 15s
55 ºC-65 ºC during 30s
72 ºC during 60s
repeat during 35 cycles
Hold at 50 °C for ever
The fist step is needed to activate the UNG enzyme, to allow it to degrade U containing ds DNA.
The second step at 95 °C will deactivate the UNG and will activate the HotGoldStar.