Why anneal at Tm? - (Dec/28/2007 )
If only half of the primers are meant to be annealed at this temperature then why isn't a lower annealing temperature used? Is preventing secondary annealing the major reason or are there others?
Typically a lower temperature *IS* used, around 4-6 degrees lower.
i use these kind of pcr protocols, for long primers or primers showing intracomplementary sequences.
these higher tm are used also in case of DMSO incompatible polymerases.