Plasmid Library Construction From Genomic DNA
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 depende...
-д хадгалсан:
| Үндсэн зохиолч: | |
|---|---|
| Бусад зохиолчид: | , , , |
| Формат: | article |
| Хэл сонгох: | англи |
| Хэвлэсэн: |
2025
|
| Нөхцлүүд: | |
| Онлайн хандалт: | https://hdl.handle.net/20.500.12381/3949 |
| Шошгууд: |
Шошго байхгүй, Энэхүү баримтыг шошголох эхний хүн болох!
|
| _version_ | 1868890204034564096 |
|---|---|
| author | Florez-Cardona, Valeria |
| author2 | Khani, Jessica McNutt, Emily Manta, Bruno Berkmen, Mehmet |
| author2_role | author author author author |
| author_browse | Berkmen, Mehmet Florez-Cardona, Valeria Khani, Jessica Manta, Bruno McNutt, Emily |
| author_facet | Florez-Cardona, Valeria Khani, Jessica McNutt, Emily Manta, Bruno Berkmen, Mehmet |
| author_role | author |
| collection | IPMON en REDI |
| dc.creator.none.fl_str_mv | Florez-Cardona, Valeria Khani, Jessica McNutt, Emily Manta, Bruno Berkmen, Mehmet |
| dc.date.none.fl_str_mv | 2025-04-22T22:26:32Z 2025-04-22T22:26:32Z 2025-01 |
| dc.identifier.none.fl_str_mv | https://hdl.handle.net/20.500.12381/3949 FCE_1_2021_1_166635 10.1002/cpz1.70088 |
| dc.language.none.fl_str_mv | eng |
| dc.publisher.none.fl_str_mv | Willey |
| dc.relation.none.fl_str_mv | https://hdl.handle.net/20.500.12381/3950 https://hdl.handle.net/20.500.12381/5242 |
| dc.rights.none.fl_str_mv | Acceso abierto info:eu-repo/semantics/openAccess Reconocimiento 4.0 Internacional. (CC BY) |
| dc.source.none.fl_str_mv | Current Protocols, Molecular Biology Section reponame:IPMON en REDI instname:Institut Pasteur de Montevideo instacron:Institut Pasteur de Montevideo |
| dc.subject.none.fl_str_mv | bacterial selection genomic DNA plasmid library construction Ciencias Naturales y Exactas Ciencias Biológicas Bioquímica y Biología Molecular |
| dc.title.none.fl_str_mv | Plasmid Library Construction From Genomic DNA |
| dc.type.none.fl_str_mv | Artículo info:eu-repo/semantics/article Aceptado info:eu-repo/semantics/acceptedVersion |
| description | 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. |
| eu_rights_str_mv | openAccess |
| format | article |
| id | anni_d6d1ef07dcc4bb72c739e5c8f17c1985 |
| identifier_str_mv | FCE_1_2021_1_166635 10.1002/cpz1.70088 |
| instacron_str | Institut Pasteur de Montevideo |
| institution | Institut Pasteur de Montevideo |
| instname_str | Institut Pasteur de Montevideo |
| language | eng |
| network_acronym_str | anni |
| network_name_str | oai-lr-anni |
| oai_identifier_str | oai:redi.anii.org.uy:20.500.12381/3949 |
| publishDate | 2025 |
| publishDateSort | 2025 |
| publisher.none.fl_str_mv | Willey |
| reponame_str | IPMON en REDI |
| repository.mail.fl_str_mv | |
| repository.name.fl_str_mv | |
| repository_id_str | |
| rights_invalid_str_mv | Acceso abierto Reconocimiento 4.0 Internacional. (CC BY) |
| spelling | Plasmid Library Construction From Genomic DNAFlorez-Cardona, ValeriaKhani, JessicaMcNutt, EmilyManta, BrunoBerkmen, Mehmetbacterial selectiongenomic DNAplasmid library constructionCiencias Naturales y ExactasCiencias BiológicasBioquímica y Biología MolecularFunctional 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.Agencia Nacional de Investigación e InnovaciónNew England Biolabs, Inc.Programa de Desarrollo de las Ciencias Basicas, Universidad de la Republica, UruguayWilley2025-04-22T22:26:32Z2025-04-22T22:26:32Z2025-01Artículoinfo:eu-repo/semantics/articleAceptadoinfo:eu-repo/semantics/acceptedVersionhttps://hdl.handle.net/20.500.12381/3949FCE_1_2021_1_16663510.1002/cpz1.70088Current Protocols, Molecular Biology Sectionreponame:IPMON en REDIinstname:Institut Pasteur de Montevideoinstacron:Institut Pasteur de Montevideoenghttps://hdl.handle.net/20.500.12381/3950https://hdl.handle.net/20.500.12381/5242Acceso abiertoinfo:eu-repo/semantics/openAccessReconocimiento 4.0 Internacional. (CC BY)oai:redi.anii.org.uy:20.500.12381/39492026-06-16T05:20:22Z |
| spellingShingle | Plasmid Library Construction From Genomic DNA Florez-Cardona, Valeria bacterial selection genomic DNA plasmid library construction Ciencias Naturales y Exactas Ciencias Biológicas Bioquímica y Biología Molecular |
| status_str | acceptedVersion |
| title | Plasmid Library Construction From Genomic DNA |
| title_full | Plasmid Library Construction From Genomic DNA |
| title_fullStr | Plasmid Library Construction From Genomic DNA |
| title_full_unstemmed | Plasmid Library Construction From Genomic DNA |
| title_short | Plasmid Library Construction From Genomic DNA |
| title_sort | Plasmid Library Construction From Genomic DNA |
| topic | bacterial selection genomic DNA plasmid library construction Ciencias Naturales y Exactas Ciencias Biológicas Bioquímica y Biología Molecular |
| url | https://hdl.handle.net/20.500.12381/3949 |