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dc.contributor.authorBjerga, Gro Elin Kjæreng
dc.contributor.authorLund, Bjarte Aarmo
dc.contributor.authorLeiros, Hanna-Kirsti S.
dc.date.accessioned2014-04-09T10:51:32Z
dc.date.available2014-04-09T10:51:32Z
dc.date.issued2014
dc.description.abstractBackground In high-throughput demanding fields, such as biotechnology and structural biology, molecular cloning is an essential tool in obtaining high yields of recombinant protein. Here, we address recently developed restriction-free methods in cloning, and present a more cost-efficient protocol that has been optimized to improve both cloning and clone screening. Results In our case study, three homologous β-lactamase genes were successfully cloned using these restriction-free protocols. To clone the genes, we chose a gene replacement strategy, where the recombinant genes contained overhangs that targeted a region of the expression vector including a cytotoxin-encoding ccdB-gene. Conclusion We provide further evidence that gene replacement can be applied with high-throughput cloning protocols. Targeting a replacement of the ccdB-gene was found to be very successful for counterselection using these protocols. This eliminated the need for treatment with the restriction enzyme DpnI that has so far been the preferred clone selection approach. We thus present an optimized cloning protocol using a restriction-free ccdB-gene replacement strategy, which allows for parallel cloning at a high-throughput level.en
dc.identifier.citationMicrobial Cell Factories 13(2014) s. 38en
dc.identifier.cristinIDFRIDAID 1122694
dc.identifier.doihttp://dx.doi.org/10.1186/1475-2859-13-38
dc.identifier.issn1475-2859
dc.identifier.urihttps://hdl.handle.net/10037/6161
dc.identifier.urnURN:NBN:no-uit_munin_5851
dc.language.isoengen
dc.publisherBioMed Centralen
dc.rights.accessRightsopenAccess
dc.subjectVDP::Mathematics and natural science: 400::Physics: 430::Space and plasma physics: 437en
dc.subjectVDP::Matematikk og Naturvitenskap: 400::Fysikk: 430::Rom- og plasmafysikk: 437en
dc.subjectVDP::Mathematics and natural science: 400::Basic biosciences: 470::Biochemistry: 476en
dc.subjectVDP::Matematikk og Naturvitenskap: 400::Basale biofag: 470::Biokjemi: 476en
dc.titleA high-throughput, restriction-free cloning and screening strategy based on ccdB-gene replacementen
dc.typeJournal articleen
dc.typeTidsskriftartikkelen
dc.typePeer revieweden


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