The light-up probe is a recently developed probe for monitoring PCR amplification in real time. It is a peptide nucleic acid (PNA) coupled to an asymmetric cyanine dye that becomes fluorescent upon binding nucleic acids. The light-up probe is used to monitor product accumulation in regular three steps PCR. It is designed to bind target DNA at annealing temperature, where the fluorescent signal is recorded, and to dissociate at elongation temperature. Here we study the effect of experimental conditions on light-up probe monitored real-time PCR. In particular, we study the effects of Mg2, primer, dNTP, Taq and probe concentrations. We find that the light-up probe can be used to monitor product formation under a wide range of conditions. Lowest threshold cycle, reflecting minimal inhibition of the PCR reaction, and large fluorescence enhancement, reflecting efficient probe binding and substantial amount of product formation, was observed in 3 mM [Mg2] 10 mM, 0.4 tM [primer], 50 iM [dNTP] 600 jiM, 0.5 U [Taqi, using 0.2 iM [light-up probe] 1 riM. Some light-up probe fluorescence enhancement is observed in non-template controls (ntc), i.e., samples containing all PCR components but template, which gives rise to primer-dimer products. This signal has a distinct shape and it reaches lower amplitudes than signals from positive samples, which makes it readily distinguishable.