Abstract
Functional genomic approaches have been effective at uncovering the function of uncharacterized genes and identifying new functions for known genes. Often these approaches rely on an in vivo screen or selection to associate genes with a phenotype of interest. These selections and screens are dependent upon the expression of proteins encoded in genomic DNA from an expression vector, such as a plasmid. Despite the utility of genomic DNA plasmid libraries, the protocols for their construction have remained unchanged in the past 40 years. Here, we present a procedure for constructing plasmid libraries from genomic DNA. This procedure is scalable and relies on simple techniques and common laboratory equipment and reagents. Briefly, the genomic DNA is extracted and then physically fragmented with a g-TUBE, overhangs are repaired, and fragments are selectively purified with magnetic beads to obtain an average fragment size of 2.5 kb. Blunted fragments are ligated into a blunt-end-digested and dephosphorylated vector. Finally, the library is amplified by electroporating the ligation into a high-transformation-efficiency Escherichia coli strain and extracting the plasmid DNA from the transformants. As a proof of concept, we built and sequenced three genomic libraries from different genomes and calculated their coverage using a next-generation sequencing (NGS) workflow. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Plasmid library construction Alternate Protocol: Selection of gDNA fragments using SageELF gel fractionator Support Protocol 1: Extraction of gDNA with phenol/chloroform Support Protocol 2: Vector preparation.