Agarose gel recovery kit

The PCR product was subjected to electrophoresis gel, and the target fragment was recovered using an agarose gel recovery kit (Tiangen, Beijing, China). The recovered product was ligated to the pGL3-Basic vector; ligation system (10 µL): 4 µL of recovered PCR product, 1 µL of vector, 5 µL of solution I. The ligation reaction was carried out at 4 °C for over 12 h. A volume of 5 µL of the ligated product was added to 100 µL DH5α cells (TransGen, Beijing, China); after heat-shock in a 42 °C water bath for 90 s, the centrifuge tube was quickly transferred to an ice bath for 1–2 min. Liquid LB medium (395 µL) was added, and the mix was shaken at 200 rpm at 37 °C for 90 min. Then, 200 µL of LB medium containing DH5α was added to AMP medium at 37 °C and cultured for 12–16 h. Single colonies were selected for culture and plasmid purification. Colony screening was performed by polymerase chain reaction (PCR) and double enzyme digestion (NheI/XhoI) experiments. The positive plasmid was sent to the Shanghai Sangon Bioengineering Company for sequencing. Plasmids with the correct sequence were amplified in large quantities and stored at −20 °C for later use.