Which extension temperature should I use, 68°C or 72°C?
Which extension temperature should I use, 68°C or 72°C?
A 68°C extension temperature is preferred for two-step PCR and when amplifying longer templates (>4 kb). This lower extension temperature dramatically improves yields of longer amplification products by reducing the depurination rate that influences amplification.
72°C should be used as the extension temperature when performing three-step standard PCR and for amplification of short fragments (<4 kb).