{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,13]],"date-time":"2026-07-13T14:05:46Z","timestamp":1783951546789,"version":"3.55.0"},"reference-count":51,"publisher":"Wiley","issue":"1","license":[{"start":{"date-parts":[[2025,1,22]],"date-time":"2025-01-22T00:00:00Z","timestamp":1737504000000},"content-version":"vor","delay-in-days":21,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Current Protocols"],"published-print":{"date-parts":[[2025,1]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>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 <jats:italic>in vivo<\/jats:italic> 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\u2010TUBE, 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\u2010end\u2010digested and dephosphorylated vector. Finally, the library is amplified by electroporating the ligation into a high\u2010transformation\u2010efficiency <jats:italic>Escherichia coli<\/jats:italic> 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\u2010generation sequencing (NGS) workflow. \u00a9 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.<\/jats:p><jats:p><jats:bold>Basic Protocol<\/jats:bold>: Plasmid library construction<\/jats:p><jats:p><jats:bold>Alternate Protocol<\/jats:bold>: Selection of gDNA fragments using SageELF gel fractionator<\/jats:p><jats:p><jats:bold>Support Protocol 1<\/jats:bold>: Extraction of gDNA with phenol\/chloroform<\/jats:p><jats:p><jats:bold>Support Protocol 2<\/jats:bold>: Vector preparation<\/jats:p>","DOI":"10.1002\/cpz1.70088","type":"journal-article","created":{"date-parts":[[2025,1,22]],"date-time":"2025-01-22T06:34:37Z","timestamp":1737527677000},"source":"Crossref","is-referenced-by-count":3,"title":["Plasmid Library Construction From Genomic DNA"],"prefix":"10.1002","volume":"5","author":[{"given":"Valeria","family":"Florez\u2010Cardona","sequence":"first","affiliation":[{"name":"New England Biolabs  Ipswich Massachusetts"},{"name":"Institut Pasteur de Montevideo  Montevideo Uruguay"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jessica","family":"Khani","sequence":"additional","affiliation":[{"name":"New England Biolabs  Ipswich Massachusetts"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Emily","family":"McNutt","sequence":"additional","affiliation":[{"name":"New England Biolabs  Ipswich Massachusetts"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bruno","family":"Manta","sequence":"additional","affiliation":[{"name":"Institut Pasteur de Montevideo  Montevideo Uruguay"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9579-9745","authenticated-orcid":false,"given":"Mehmet","family":"Berkmen","sequence":"additional","affiliation":[{"name":"New England Biolabs  Ipswich Massachusetts"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2025,1,22]]},"reference":[{"key":"e_1_2_10_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0378-1119(97)00044-9"},{"key":"e_1_2_10_3_1","doi-asserted-by":"publisher","DOI":"10.1038\/nbt890"},{"key":"e_1_2_10_4_1","doi-asserted-by":"publisher","DOI":"10.1002\/bip.22191"},{"key":"e_1_2_10_5_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0153158"},{"key":"e_1_2_10_6_1","doi-asserted-by":"publisher","DOI":"10.1111\/1751-7915.13265"},{"key":"e_1_2_10_7_1","doi-asserted-by":"publisher","DOI":"10.4014\/jmb.1212.12008"},{"key":"e_1_2_10_8_1","first-page":"8","article-title":"Cloning of \u03bb DNA fragments into pUC19 vector to study the ligation efficiency of NdeI\u2010digested and HindII\u2010digested pUC19 by T4 DNA ligase","volume":"8","author":"Chow P.","year":"2005","journal-title":"Journal of Entrepreneurship, Management, and Innovation"},{"key":"e_1_2_10_9_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.RA118.003302"},{"key":"e_1_2_10_10_1","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.02169-09"},{"key":"e_1_2_10_11_1","unstructured":"Covaris. (2020). g\u2010TUBE for DNA shearing. M020006_Rev_Jan2020."},{"key":"e_1_2_10_12_1","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2021.723874"},{"key":"e_1_2_10_13_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0065956"},{"key":"e_1_2_10_14_1","first-page":"S36","article-title":"Complementary DNA shearing and size selection tools for mate\u2010pairs library construction","volume":"23","author":"Durin G.","year":"2012","journal-title":"Journal of Biomolecular Techniques"},{"key":"e_1_2_10_15_1","doi-asserted-by":"publisher","DOI":"10.1186\/s13068-022-02172-4"},{"key":"e_1_2_10_16_1","doi-asserted-by":"publisher","DOI":"10.1038\/nprot.2007.204"},{"key":"e_1_2_10_17_1","doi-asserted-by":"publisher","DOI":"10.1186\/s12934-014-0105-4"},{"key":"e_1_2_10_18_1","doi-asserted-by":"publisher","DOI":"10.1002\/0471142727.mba03ds76"},{"issue":"10","key":"e_1_2_10_19_1","article-title":"Single\u2010stranded DNA library preparation from highly degraded DNA using T4 DNA ligase","volume":"45","author":"Gansauge M. T.","year":"2017","journal-title":"Nucleic Acids Research"},{"key":"e_1_2_10_20_1","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-60761-823-2_19"},{"key":"e_1_2_10_21_1","volume-title":"Formulation of cloning strategies","author":"Green J.","year":"1998"},{"key":"e_1_2_10_22_1","doi-asserted-by":"publisher","DOI":"10.1101\/pdb.top100768"},{"key":"e_1_2_10_23_1","doi-asserted-by":"publisher","DOI":"10.1101\/pdb.prot100669"},{"key":"e_1_2_10_24_1","doi-asserted-by":"publisher","DOI":"10.1016\/S1074-5521(98)90108-9"},{"key":"e_1_2_10_25_1","doi-asserted-by":"publisher","DOI":"10.1016\/0003-9861(61)90016-9"},{"key":"e_1_2_10_26_1","doi-asserted-by":"publisher","DOI":"10.1186\/1756-0500-3-291"},{"key":"e_1_2_10_27_1","doi-asserted-by":"publisher","DOI":"10.1186\/2193-1801-2-441"},{"issue":"1","key":"e_1_2_10_28_1","first-page":"28","article-title":"An efficient method for blunt\u2010end ligation of OCR products","volume":"12","author":"Liu Z. G.","year":"1992","journal-title":"Biotechniques"},{"key":"e_1_2_10_29_1","doi-asserted-by":"publisher","DOI":"10.3390\/mps6050089"},{"key":"e_1_2_10_30_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.heliyon.2024.e26996"},{"key":"e_1_2_10_31_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-019-42868-6"},{"key":"e_1_2_10_32_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0150528"},{"key":"e_1_2_10_33_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.RA120.016350"},{"key":"e_1_2_10_34_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41564-024-01687-w"},{"key":"e_1_2_10_35_1","doi-asserted-by":"publisher","DOI":"10.1093\/clinchem\/46.11.1851"},{"issue":"5","key":"e_1_2_10_36_1","article-title":"BiG\u2010MAP: An automated pipeline to profile metabolic gene cluster abundance and expression in microbiomes","volume":"6","author":"Pascal A. V.","year":"2021","journal-title":"mSystems"},{"key":"e_1_2_10_37_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/11.22.7853"},{"key":"e_1_2_10_38_1","doi-asserted-by":"publisher","DOI":"10.1021\/acssynbio.8b00333"},{"key":"e_1_2_10_39_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.chembiol.2023.10.001"},{"key":"e_1_2_10_40_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/25.11.2106"},{"key":"e_1_2_10_41_1","volume-title":"Molecular cloning: A laboratory manual","author":"Sambrook J.","year":"1989"},{"key":"e_1_2_10_42_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.4103"},{"key":"e_1_2_10_43_1","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-4939-6691-2_1"},{"key":"e_1_2_10_44_1","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-4939-6691-2_16"},{"key":"e_1_2_10_45_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.apsoil.2015.04.005"},{"key":"e_1_2_10_46_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.sbi.2022.102416"},{"key":"e_1_2_10_47_1","doi-asserted-by":"publisher","DOI":"10.1038\/nprot.2006.90"},{"issue":"4","key":"e_1_2_10_48_1","first-page":"408","article-title":"Computational methods for discovering gene networks from expression data","volume":"10","author":"Wei\u2010Po L.","year":"2009","journal-title":"Briefings in Bioinformatics"},{"key":"e_1_2_10_49_1","doi-asserted-by":"publisher","DOI":"10.1016\/0022-2836(79)90270-5"},{"key":"e_1_2_10_50_1","doi-asserted-by":"publisher","DOI":"10.1093\/protein\/gzt024"},{"key":"e_1_2_10_51_1","doi-asserted-by":"publisher","DOI":"10.15252\/msb.20145866"},{"key":"e_1_2_10_52_1","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2018.02132"}],"container-title":["Current Protocols"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cpz1.70088","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,7,24]],"date-time":"2025-07-24T15:22:31Z","timestamp":1753370551000},"score":1,"resource":{"primary":{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/10.1002\/cpz1.70088"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,1]]},"references-count":51,"journal-issue":{"issue":"1","published-print":{"date-parts":[[2025,1]]}},"alternative-id":["10.1002\/cpz1.70088"],"URL":"https:\/\/doi.org\/10.1002\/cpz1.70088","archive":["Portico"],"relation":{},"ISSN":["2691-1299","2691-1299"],"issn-type":[{"value":"2691-1299","type":"print"},{"value":"2691-1299","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,1]]},"article-number":"e70088"}}