NEB® has developed a protocol using NEBuilder HiFi DNA Assembly Master Mix to simplify the construction of multiple site-directed mutagenesis. The technique involves the design of complimentary flanking primers to align fragments. This application note describes the use of the NEBuilder HiFi DNA Assembly Master Mix to generate multiple site-directed mutagenesis at the same time.
We recommend 18-20nt overlaps but the length of the primers can vary. The primers are completely overlapping as illustrated below. The goal is to achieve balanced Ta’s for the primer pairs. The mutations are positioned in the middle of the primer. There must be enough bases on either side, and most importantly 3’ of the mutation, so that the primer will anneal properly to the template.