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Updated: Aug 8, 2025

A Customizable Protocol for String Assembly gRNA Cloning STAgR
Published on: December 26, 2018
Assembling Multiple Fragments: The Gibson Assembly
1Centre for Enzyme Innovation, University of Portsmouth, Portsmouth, UK. luisana.avilan@port.ac.uk.
Abstract:
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.
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