000125930 001__ 125930 000125930 005__ 20240731103401.0 000125930 0247_ $$2doi$$a10.1007/978-1-0716-3004-4_3 000125930 0248_ $$2sideral$$a133348 000125930 037__ $$aART-2023-133348 000125930 041__ $$aeng 000125930 100__ $$0(orcid)0000-0003-3785-298X$$aArroyo-Urea, Sandra$$uUniversidad de Zaragoza 000125930 245__ $$aMolecular cloning using in vivo DNA assembly 000125930 260__ $$c2023 000125930 5060_ $$aAccess copy available to the general public$$fUnrestricted 000125930 5203_ $$aHere we describe the in vivo DNA assembly approach, where molecular cloning procedures are performed using an E. coli recA-independent recombination pathway, which assembles linear fragments of DNA with short homologous termini. This pathway is present in all standard laboratory E. coli strains and, by bypassing the need for in vitro DNA assembly, allows simplified molecular cloning to be performed without the plasmid instability issues associated with specialized recombination-cloning bacterial strains. The methodology requires specific primer design and can perform all standard plasmid modifications (insertions, deletions, mutagenesis, and sub-cloning) in a rapid, simple, and cost-efficient manner, as it does not require commercial kits or specialized bacterial strains. Additionally, this approach can be used to perform complex procedures such as multiple modifications to a plasmid, as up to 6 linear fragments can be assembled in vivo by this recombination pathway. Procedures generally require less than 3 h, involving PCR amplification, DpnI digestion of template DNA, and transformation, upon which circular plasmids are assembled. In this chapter we describe the requirements, procedure, and potential pitfalls when using this technique, as well as protocol variations to overcome the most common issues. 000125930 540__ $$9info:eu-repo/semantics/openAccess$$aby$$uhttp://creativecommons.org/licenses/by/3.0/es/ 000125930 592__ $$a0.399$$b2023 000125930 593__ $$aGenetics$$c2023$$dQ3 000125930 593__ $$aMolecular Biology$$c2023$$dQ4 000125930 594__ $$a2.0$$b2023 000125930 655_4 $$ainfo:eu-repo/semantics/article$$vinfo:eu-repo/semantics/acceptedVersion 000125930 700__ $$aWatson, Jake F. 000125930 700__ $$0(orcid)0000-0002-4254-3148$$aGarcía-Nafría, Javier$$uUniversidad de Zaragoza 000125930 7102_ $$11002$$2060$$aUniversidad de Zaragoza$$bDpto. Bioq.Biolog.Mol. Celular$$cÁrea Bioquímica y Biolog.Mole. 000125930 773__ $$g2633 (2023), 33-44$$pMethods mol. biol. (Clifton N.J.)$$tMethods in Molecular Biology$$x1064-3745 000125930 8564_ $$s282030$$uhttps://zaguan.unizar.es/record/125930/files/texto_completo.pdf$$yPostprint 000125930 8564_ $$s1657105$$uhttps://zaguan.unizar.es/record/125930/files/texto_completo.jpg?subformat=icon$$xicon$$yPostprint 000125930 909CO $$ooai:zaguan.unizar.es:125930$$particulos$$pdriver 000125930 951__ $$a2024-07-31-09:59:20 000125930 980__ $$aARTICLE