Gibson Assembly®

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.

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Benefits of the Gibson Assembly

  • Increased number of successful assembly products, particularly for longer or greater number of fragments
  • Flexible sequence design (scarless cloning)
  • No clean-up step required
  • Complex assembly achieved in 1 hour
  • DNA can be used immediately for transformation, or as template for PCR or RCA
  • Easily adapted for multiple DNA manipulations, including site directed mutagenesis, insertions and deletions

Benefits of the Gibson Assembly Cloning Kit

  • Assembly and transformation in just under two hours
  • Flexible sequence design (scar-less cloning)
  • No PCR clean-up step required
  • High transformation efficiencies for inserts up to 20 kb
  • Easily adapted for multiple DNA manipulations, including site-directed mutagenesis
  • Includes competent cells


  1. Gibson, D.G. et al. (2009) Nature Methods, 343–345.
  2. Gibson, D.G. et al. (2010) Nature Methods, 901–903.

Here is what Customers Are Saying About Gibson Assembly

“It is very convenient and fast to clone a gene of interest into a large vector, in which unique restriction sites are difficult to find or manipulate.”
Researcher, Washington University

“The Gibson Master Mix is a superior cloning tool that inevitably saves large blocks of time, increases work flow, eliminates false positive clones, and saves a great deal of money! Compared to traditional cloning, the elimination of having to produce multiple intermediate clones for multiple fragment ligations is an enormous time saver. Furthermore, the fact that it works the first time, every time saves you from the frustrating and time consuming troubleshooting steps. I easily saved a week’s worth screening and subcloning with this mix. My approach to cloning anything is forever changed.”
Researcher; Sanford-Burnham Medical Research Institute

Overview of the Gibson Assembly Cloning Method

Some components of this product are manufactured by New England Biolabs, Inc. under license from Synthetic Genomics, Inc.