When would it be appropriate to try alternative cycling conditions for KASP?

The standard cycling conditions for KASP are the 61-55 ºC touchdown protocol (click for protocol).  There are two additional KASP thermal cycling protocols that may either be recommended as optimal (for laboratory-validated and Assay Search Tool assays) or may be worth trying (for design-only assays) if non-optimal results are obtained with the standard 61-55 ºC touchdown protocol.

The ‘68-62 ºC touchdown protocol’ (click for protocol)  may be beneficial for assays that have a high %GC content (greater than 65%).

The ‘2-step 57 ºC protocol’ (click for protocol) may be beneficial for assays that have a low %GC content (less than 40%).

Full details of all KASP thermal cycling conditions can be found in the ‘KASP thermal cycling conditions’(click for document).