title: A gel-less isolation of untagged plasmid DNA insert from vector backbone in homogeneous format.
abstract: Agarose gel electrophoresis with subsequent DNA extraction from gel is routinely used for DNA fragment isolation after plasmid DNA digestion. We describe a gel-less method for DNA fragment isolation after plasmid DNA digestion which is based on in-solution negative selection through depletion of vector backbone bearing LoxP sites by sorption on solid phase-immobilized mutated Cre recombinase. The method might be especially useful in preparation of DNA fragments for transgenic animal generation where residual agarose presence is a concern, and DNA fragments are frequently large in size and thus might be mechanically damaged during purification with conventional affinity-based gel extraction methods.
authors.affiliation: Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilov street, Moscow 119334, Russia.
authors.forename: Svetlana V
authors.initials: SV
authors.lastname: Kalinichenko
authors.affiliation: Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilov street, Moscow 119334, Russia.
authors.forename: Mikhail V
authors.initials: MV
authors.lastname: Shepelev
authors.affiliation: Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilov street, Moscow 119334, Russia. Electronic address: igorvk@igb.ac.ru.
authors.forename: Igor V
authors.initials: IV
authors.lastname: Korobko
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D005822:Genetic Vectors; D019426:Integrases / Q000235:genetics*; D010957:Plasmids / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D004247:DNA; C045073:Cre recombinase; D019426:Integrases
keywords: DNA fragment isolation; Homogeneous format; Negative selection; Plasmid DNA digestion