Some people say keep the overlapping size at 40-45 bp, but this way the annealing temperature will be very high (nearly 81C). I want to keep it at 21 bp, meaning desining two primers that are exactly complementary of each other (Reverse primer of Fragment 1 to be complementary to Forward primer or fragment 2). should I worry about the length at all? or they will bind anyway?
Edited by Curtis, 09 April 2012 - 10:04 PM.