FAQ: What are the longest overlaps that can be used with this method?

Both the quantity of 5´ exonuclease in the NEBuilder HiFi DNA Assembly Master Mix and a 15 minute assembly reaction time have been optimized for the assembly of DNA molecules with ≤ 25 bp overlaps. If assembly reaction time is increased to 60 minutes, overlaps up to 40 bp may be used with the NEBuilder HiFi DNA Assembly Master Mix.