Screening of LoxP-positive recombinant clones by self-primer colony polymerase chain reaction

Objective To investigate the screening and evaluating methods of positive recombinant clones for small fragments such as LoxP sequence. Methods Synthesized LoxP and vector complementary sequence were used as the upper and lower primer respectively, and colonies were used directly as the templates of polymerase chain reaction (PCR). The presence of 784 bp strap in electrophoresis was seen as positive. The positive recombinant clones screened by PCR were evaluated contrastively by restriction endonuclease digestion and verified by DNA sequence analysis. Results Among the six colonies randomly screened by PCR, three showed positive straps and one was verified by DNA sequence analysis. However, the electrophoresis only showed unclear and clouding straps when the three positive recombinant clones were evaluated by restriction endonuclease digestion. Conclusion Self-primer colony PCR is a high-speed, convenient, economic and effective method for screening and evaluating of positive clones recombinated by small fragments such as LoxP sequence.