FAQ: How does overlap length affect NEBuilder HiFi DNA Assembly efficiency?

Longer overlaps (20-30 base pairs) increase assembly efficiency.

Four fragments (~20 fmol) with 15 or 20 bp overlap were assembled using NEBuilder HiFi DNA Assembly Master Mix (NEB #E2621) at 50°C for 60 minutes to create a pUC19 vector. 2µl of assembled mix was transformed into NEB 5-alpha Competent E. coli (NEB #C2987) and spread on an LB/Amp plate containing IPTG and X-gal. Blue colonies that indicated correct assembly were counted. NEBuilder HiFi DNA Assembly Mix is more efficient using a 20 bp overlap compared to a 15 bp overlap.