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.
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    Primer Design and Fragment Assembly Using NEBuilder HiFi DNA Assembly or Gibson Assembly®

    Watch an interactive tutorial on primer design to see how simple it really is to clone with either NEBuilder HiFi DNA Assembly or the Gibson Assembly Cloning Kit.