Is it possible to perform a UNG treatment when using QuantiTect kits?
Yes - dTTP in the QuantiTect Master Mix in QuantiTect SYBR Green and Probe PCR Kits is partially substituted by dUTP, therefore allowing treatment with uracil-N-glycosylase.
However, only heat-labile UNG can be used for one-step RT-PCR with the QuantiTect Probe RT-PCR and SYBR Green RT-PCR Kits. This is because heat stable UNG (isolated from E. coli) will destroy any cDNA synthesized during the RT step at 50°C. Heat-labile UNG will have lost any activity after a few minutes of the RT-step and therefore cannot interfere with cDNA synthesis.
For heat-stable uracil-N-glycosylase, please use the QuantiTect SYBR Green PCR +UNG (cat. no. 204163) or QuantiTect Probe PCR +UNG Kits (cat. no. 204363). Both kits include heat-stabile uracil-N-glycosylase. Although QIAGEN UNG is inactivated by incubation at 95°C for 15 minutes, the enzyme may exhibit residual activity at lower temperatures due to refolding. Therefore, it is recommended to perform subsequent PCR using a temperature of 55°C or above for the annealing step.