Optimized generation of vectors for the construction of Haloferax volcanii deletion mutants.
A method for the construction of in frame deletions in chromosomal genes of the archaeon Haloferax volcanii has been described recently (Allers et al. (2004), Appl. Environ. Microbiol. 70:943-953). Two steps of the procedure for deletion vector construction were optimized. First, the deletion version of the gene of interest was generated by fusion of two PCR products, which were comprised of the upstream and downstream regions of the gene, respectively, using a third PCR reaction. Second, the fusion PCR product was ligated with the optimized vector pMH101 in the presence of one of four possible restriction enzymes ("restriction selection cloning"). Taken together, the optimized procedure omits one cloning step and enhances both speed and efficiency of deletion vector construction considerably.
Hammelmann
M
M
Institute for Molecular Biosciences, Biocentre, Goethe-University, D-60438 Frankfurt, Germany.
Soppa
Jörg
J
D015252:Deoxyribonucleases, Type II Site-Specific; D017353:Gene Deletion; D005821:Genetic Techniques; D005822:Genetic Vectors / Q000235:genetics*; D019622:Haloferax volcanii / Q000235:genetics*; D009154:Mutation; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D019642:RNA, Archaeal / Q000235:genetics; D015183:Restriction Mapping
D019642:RNA, Archaeal; C064433:endodeoxyribonuclease SnaBI; D015252:Deoxyribonucleases, Type II Site-Specific