PCR amplification was conducted using Hieff Canace® PCR Master Mix (Yeasen Biotechnology Co., Ltd.). Molecular cloning employed the TSINGKE TSV-007VS pClone007 Versatile Simple Vector Kit (Tsingke Biotechnology Co., Ltd., Beijing, China) with TreliefTM 5α Chemically Competent Cell (Tsingke Biotechnology Co., Ltd.). Plasmid purification and DNA gel extraction were performed using high-purity kits (Gensand).|||The VmCarEs-6 gene sequence was identified through comparative analysis of transcriptomic data from stress-exposed T. trifolii (unpublished data). Gene-specific primers were designed using Premier 5 software: VmCarEs-F (5′-CTTGATGATTCCTTGTC-3′) and VmCarEs-R (5′-TGGTGGG GTAAAACTA-3′), synthesized by Tsingke Biotechnology Co., Ltd. (Beijing). PCR amplification was performed in a 50 µL reaction mixture containing 2 µL cDNA template, 25 µL Hieff Cana ce® PCR Master Mix, 2 µL each of forward and reverse primers, and19µL nuclease-free water.
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