Figure 2

One-step cloning by homologous recombination in yeast of an ACLSV FL-cDNA under the control of T7 promoter. (A) 0.8% non-denaturing agarose gel electrophoresis of the two PCR fragments used to transform yeast cells. Lane 1: PCR product corresponding to the Yeast-PBS70T amplified using the divergent T7ACR/30Anotvec primer pair; Lane 2: ACLSV T7-FL-cDNA amplified using the FLAC5/FLAC3 primer pair; (M): Molecular mass marker (Invitrogen 1 kb ladder). (B) Schematic representation of the cloning by homologous recombination strategy. The 30 bp overlapping regions between the two PCR fragments in which homologous recombination takes place are indicated. (C) Bgl II restriction analysis (lanes 1-4) of four pools of 10 independent recombinant plasmids recovered following retransformation of E. coli cells using the plasmid preparation purified from the bulked transformant yeast cells, (M): Molecular mass markers (Invitrogen1 kb ladder). (D) Analysis by non-denaturing 1% agarose gel electrophoresis of the same four pools of plasmids, following linearization with Not I (lanes 1-4), (M): Molecular mass markers (Invitrogen 1 kb ladder). (E) Analysis by 1% denaturing agarose gel electrophoresis of the in vitro RNA transcripts synthesized from the Not I-linearized plasmid pools, (M): 0.5-10 kb RNA ladder (Sigma).