HomeFAQsWhat are the shortest overlaps that can be used with this assembly method?
FAQ: What are the shortest overlaps that can be used with this assembly method?
Productive assembly has been achieved for DNA fragments with as little as a 12 bp overlap, however, it depends on the GC content of the overlap. We recommend using at least 15 bp overlaps, or more, for dsDNA assembly with a Tm ≥ 48°C (AT pair = 2°C and GC pair = 4°C). Increasing the length of overlap between fragments also reduces the amount of DNA needed for 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.
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