Gibson Assembly® was developed by Dr. Daniel Gibson and his colleagues at the J. Craig Venter Institute and licensed to NEB by Synthetic Genomics, Inc. The technique allows for the successful assembly of multiple DNA fragments, regardless of fragment length or end compatibility. The Gibson Assembly Cloning Kit (NEB #E5510) combines the power of Gibson Assembly Master Mix (NEB #E2611) with NEB 5-alpha Competent E. coli (NEB # C2987), enabling fragment assembly and transformation in just over an hour. Gibson Assembly efficiently joins multiple overlapping DNA fragments in a single-tube isothermal reaction (1,2). The Gibson Assembly Master Mix includes three different enzymatic activities that perform in a single buffer: The exonuclease creates a single-stranded 3´ overhang that facilitates the annealing of fragments that share complementarity at one end. The polymerase fills in gaps within each annealed fragment. The DNA ligase seals nicks in the assembled DNA. The assembled, fully-sealed construct is then transformed into NEB 5-alpha Competent E. coli. The entire protocol, from assembly to transformation, takes a little over an hour.