pHP45omega-Km

[2] describes a procedure combining the advantages of in vitro DNA linker mutagenesis with those of in vivo transposition mutagenesis. Insert the symmetrical 2.0-kb omega fragment, with an antibiotic resistance gene flanked by short inverted repeats carrying translation termination signals and synthetic polylinkers, into a linearized plasmid. The omega fragment terminates RNA and protein synthesis prematurely, allowing the definition and mapping of both transcription and translation units. Because of its symmetry, it can be inserted in either direction. Cut pHP45omega with BssHII + SphI, removing the spcR/strR gene, and make the ends blunt. Ligate this to a blunt-end Tn5 1.5-kb HindIII-SalI fragment, containing the kanamycin resistance gene. [1] Because resistance levels may vary, it is important to determine the proper antibiotic concentration for each strain or isolate. [1] This is one of a series (ATCC37653-ATCC37657) of vectors for in vitro mutagenesis (via the omega interposon) of gram-negative bacteria. [1] Restriction digests of the clone give the following sizes (kb): EcoRI–2.35, 2.35; HindIII–2.4, 2.3; SalI–4.7. (ATCC staff) Medium is 1227 LB plus ampicillin. NCBI gi: 209010 Hosts: E.coli C600, broad host range, E.coli. Related vectors: pBR322, pKTH604, R100.1 omega element, pHP45omega-Cm, pHP45omega-Hg, pHP45omega-Tc.