title: Rapid and efficient subcloning of DNA without dephosphorylation or gel electrophoresis.
abstract: Conventional subcloning into plasmid vectors often involves dephosphorylation, gel electrophoresis, DNA extraction, and purification to isolate the target insert and the cleaved plasmid. This is not only time-consuming but very often problematic. We have developed strategies that can circumvent these steps by mixing digested donor and recipient plasmids together for ligation. These strategies capitalizes on: (1) the ability to enhance the ligation efficiency of desired DNA fragments into the target vector by the generation and removal of small (< 50 bp) fragments from nontarget DNA using peripheral restriction sites and spin column technology and (2) the elimination of undesired ligation products after ligation by using the Lac Z gene, differences in antibiotic resistance among plasmid vectors, and unique restriction sites situated in nontarget DNA fragments.
authors.affiliation: Veterinary Science, 341 Gluck Equine Research Center, University of Kentucky, Lexington, KY, USA.
authors.forename: Qilong
authors.initials: Q
authors.lastname: Xu
authors.forename: Deqing
authors.initials: D
authors.lastname: Zhang
authors.forename: Bruce
authors.initials: B
authors.lastname: Downie
mesh_terms: D002845:Chromatography / Q000379:methods*; D003001:Cloning, Molecular / Q000379:methods*; D020552:Comet Assay; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D005818:Genetic Engineering / Q000379:methods*; D010766:Phosphorylation; D013048:Specimen Handling / Q000379:methods*
chemical_list: D004247:DNA; D004262:DNA Restriction Enzymes