Figure 6

The SMLP method can be used for mutagenesis of the plasmids up to 17.3 kb. (A) A diagram showing the pLV-FLNA plasmid and the positions of the PCR primers designed. The primers were included in Table S2. (B) Analysis of the small and large DNA fragments from PCR by agarose gel electrophoresis. PCR I and PCR II were performed using the primer pair of pLVFP and D1159ARP and the primer pair of pLVRP and D1159AFP, respectively, where the pLV-FLNA plasmids acted as DNA templates. PCR products were detected by agarose gel electrophoresis as for Fig. 5B. (C) An image showing the result of transformation. A transformation assay was performed as described in Fig. 3D. (D) Analysis of the plasmids from site-directed mutagenesis by agarose gel electrophoresis. Plasmid detection was performed as for Fig. 3E. (E) The result of DNA sequencing for the pLV-FLNA-D1159A mutant. The left panel is the DNA sequencing map of the pLV-FLNA-D1159A mutant, where the mutated base is framed by the red rectangle lines. The right panel is the original and mutated DNA fragments presented as for Fig. 5E.