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I'm having problems with primer efficiency. The primers I use amplify a product of 300pb. I have used the reaction protocol I have used before with 100pb product. I usually do 65ºC for 1 min for annealing and 95ºC for 1 min for melting. Should I add an extension step in the reaction? or maybe optimize dNTP concentration?


I do not think there is a big difference between 100bp and 300 bp, unless GC contents have a big difference between two amplicons. Try add one extension step. Good luck