Assembling multiple fragments: the Gibson Assembly

Luisana Avilan

Research output: Chapter in Book/Report/Conference proceedingChapter (peer-reviewed)peer-review


The Gibson Assembly is a popular method for molecular cloning which has been developed specifically to join several fragments together in a specific order, without the constraint of restriction enzyme sites. This method is based on the assembly of overlapping fragments, generally produced by PCR, and then combining them using three enzymes: a 5′ exonuclease, a DNA polymerase, and a DNA ligase, in an isothermal reaction. Here, we describe this method, including the design of primers for the generation of the overlapping fragments and the assembly; to this end, we provide an example involving joining two fragments in a single plasmid.
Original languageEnglish
Title of host publicationDNA Manipulation and Analysis
EditorsGarry Scarlett
Place of PublicationNew York
PublisherHumana Press
Number of pages9
ISBN (Electronic)9781071630044
ISBN (Print)9781071630037
Publication statusPublished - 1 Mar 2023

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029


  • Gibson Assembly
  • cloning
  • overlapping primers

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