On 2014 Feb 12, Stephen Bond commented:

Users may find that their projects have a higher success rate if they switch from the 3-step secondary PCR reaction (as described in the paper) to a 2-step reaction, where the annealing step is essentially merged with the 72°C extension step (setting hold time to 30 sec/kB). This has been validated in our lab, and also by a number of independent investigators who have shared their experience through personal correspondence.

On 2014 Feb 12, Stephen Bond commented:

Users may find that their projects have a higher success rate if they switch from the 3-step secondary PCR reaction (as described in the paper) to a 2-step reaction, where the annealing step is essentially merged with the 72°C extension step (setting hold time to 30 sec/kB). This has been validated in our lab, and also by a number of independent investigators who have shared their experience through personal correspondence.