Table 2. LightCycler experimental conditions

The Lightcycler protocol consists of four programs, each involving one or more incubation period or segment. In the first UDG Denaturation program, incubation at 50 °C for 120 s enables uracil-DNA glycosylase (UDG) to destroy any contaminating dU containing PCR product from previous reactions. Following denaturation at 95 °C, forty cycles of amplification are performed. The temperature transition rate between segments is 20 °C/s throughout, except between segments 2 and 3 of the melting program when the temperature is slowly raised at a rate of 0.1 °C/s to enable accurate discrimination between mutant and wild type alleles.

  Program Cycles        Segment 1         Segment 2        Segment 3
------------------   ---------------   ---------------   -------------
UDG Denaturation 1   50 °C for 120 s   95 °C for 120 s
Amplification 40     95 °C for   5 s   50 °C for   5 s   72 °C for 5 s
Melting 1            95 °C for  30 s   40 °C for 120 s   95 °C for 0 s
Cooling 1            40 °C for  30 s