When would it be appropriate to try alternative cycling conditions for KASP?
The standard cycling conditions for KASP are the 61-55ºC 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 61-55ºC touchdown protocol.
The ‘68-62ºC touchdown protocol’ (click for protocol) may be beneficial for assays that have a high %GC content (greater than 65%).
The ‘2-step 57ºC 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).