Easytaq pcr supermix

The standards of enrofloxacin, chloramphenicol, ampicillin, trimethoprim, and sulfamethoxazole were purchased from Dr. Ehrenstorfer (Germany). Ciprofloxacin standard was obtained from MedChemExpress (New Jersey, United States). Tetracycline standard was from China Institute of Veterinary Drug Control (Beijing, China), and 2 × EasyTaq PCR SuperMix and Phanta super-fidelity DNA Polymerase were purchased from Vazyme (Nanjing, China). The pUCm-T vector, IPTG, and X-Gal were from Beyotime Biotechnology (Nantong, China). The primers of this study were all synthesized by Genscript (Nanjing, China).

An assembled unigene (Unigene5167) of 657 bp annotated as Df from our previous transcriptome data [45 ] was selected for further cloning of the full-length cDNA of CiDf. Two specific primers, Df 5′ and Df 3′ (Table 1), were designed to amplify the full-length cDNA of CiDf by RACE technology. The total volume of the PCR was 50 µL, including 1 µL of cDNA, 25 µL of 2× EasyTaq PCR SuperMix (Vazyme, Nanjing, China), 1 µL of each gene-specific primer Df 5′ (Df 3′), 1 µL of Universal Primer A Mix, and 22 µL of H₂O. The PCR programs were run as follows: 5 cycles at 94 °C for 30 s, 72 °C for 3 min; 5 cycles at 94 °C for 30 s, 70 °C for 30 s, and 72 °C for 3 min; 25 cycles at 94 °C for 30 s, 68 °C for 30 s, and 72 °C for 3 min. The PCR products were gel-purified using the MiniBest Agrose Gel DNA Extraction Kit Ver. 4.0 (Takara, Kyoto, Japan), cloned into the pUCm-T vector (Kanglang, Shanghai, China), and then sequenced by M13F(-47) and RV-M primers (Table 1).