Figure 3

The SMLP method can be used for mutagenesis of the plasmid about 13.3 kb. (A) A diagram showing the pcDNA-FLNA plasmid and the primers designed for sit-directed mutagenesis. pcDNAFP represents a pcDNA plasmid forward primer; pcDNARP represents a pcDNA plasmid reverse primer. M102VFP is an M102V forward primer; M102VRP is an M102V reverse primer. The red line represents the mutated site in the plasmid. (B) Analysis of the small DNA fragments from PCR by agarose gel electrophoresis. PCR was performed using the pcDNA-FLNA plasmid as DNA templates, the primer pair of pcDNAFP and M102VRP, and Phanta Max Super-fidelity DNA polymerase. PCRSF-M102V represents the PCR small fragments for the mutagenesis of M102V. (C) Analysis of the large DNA fragments from PCR by agarose gel electrophoresis. PCR was performed as in (B) using the primer pair of pcDNARP and M102VFP. PCRLF-M102V represents the PCR large fragments for the mutagenesis of M102V. (D) The result of the transformation with 3μL ligation samples for the mutagenesis of M102V. The transformation plate was pictured using an EOS digital camera (Canon). (E) Detection of the plasmids from the mutagenesis of M102V by agarose gel electrophoresis. DNA plasmids were prepared using a Miniprep kit (CWBiol, China), and were subjected to detection with agarose gel electrophoresis. (F) The result of DNA sequencing for the M102V mutant. The DNA sequencing map for the M102V mutant was presented in the upper panel in which a mutated base was framed by the red rectangle lines. Both original and mutated DNA fragments were presented in the bottom panel, where the bases displayed in the sequencing map were outlined by the red rectangle lines. The original and mutated bases were presented in green and red colours, respectively.