Amplification of the DNA of pBAC/oriV clones carrying (A,C) 108-kb or (B) 122-kb inserts of foreign DNA, when propagated in the DH10B host and in two commercial hosts, GeneHogs (Invitrogen) and Stbl2 (Life Technologies, presently Invitrogen). All three host strains contain thearaC–P BAD–trfA254 cassette at theirattB λ site. Growth conditions and induction of TrfA synthesis by L-arabinose (A) are described in Methods. DNA samples prepared by phenol extraction and ethanol precipitation were digested with SalI (A,C) or with SmaI (B) and run on a 0.6% agarose gel. (A) Amplification of pBAC/oriV carrying a 108-kb insert (pCG275) in the JW427 host (trfA254; see JW439 in Table 2). (Lane 1) LB medium (LB); (lane 2) LB + 0.01% A. (B) Amplification of pBAC/oriV carrying a 122-kb insert (pCG274) in JW480 (GeneHogstrfA254). (Lane 1) LB; (lane 2) LB + 0.01% A. (C) Amplification of pBAC/oriV carrying a 108-kb insert (pCG275) in JW526 (Stbl2 trfA254). (Lane1) LB; (lanes 2–6) LB + 0.01% A. (Lanes3–6) DNA preparations were diluted 1/2, 1/4, 1/10, and 1/20, respectively. Comparison of lanes 1 and 6indicates an ∼30-fold amplification of the plasmid with the 108-kb DNA insert.
