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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?

Cheers, Rob


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.