Thermocycling program for this pair of primers consists of 40 cycles of 95°C for 15s and 60°C for 1 min with an initial cycle of 95°C for 1 min. Accumulations of the PCR products are detected in each cycle by monitoring the increase in fluorescence of the reporter dye double-strand DNA-binding SYBR® Green.
Dissociation curve (melting curve, Tm) analysis should be performed within the temperature range of 60°C to 95°C to detect specific amplification of the target sequence. GeneAmp® 5700 SDS software (PE Applied Biosystems) can be used for melting curve analysis. Details of melting analysis are provided in Chapter 9.
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