Takara minibest bacteria genomic dna extraction kit

The viral genomic DNA or RNA was extracted using the QlAamp Viral DNA/RNA Mini Kit (QIAGEN, Hilden, Germany). RNAs were reverse-transcribed using the PrimeScript™ RT Reagent Kit (TaKaRa BIO Inc., Dalian, China). Salmonella, Pm, MRSA, HPS, and S. suis were cultured, and the genomic DNA was extracted using TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit (TaKaRa). All DNA and cDNA were stored at −20°C.
The target genes of the nine porcine viral pathogens were amplified using their specific primer pair. The amplified products were purified using the TaKaRa MiniBEST Agarose Gel DNA Extraction Kit (TaKaRa BIO Inc., Dalian, China). The purified DNA was ligated into the pMD19-T vector, and the ligated constructs were transformed into E. coli DH5a cells cultured in the presence of ampicillin (100 μg/mL). The recombinant plasmid construct was confirmed by DNA sequencing (Life Technologies Inc., Shanghai, China), and the sequence data were analyzed using DNASTAR software and compared with the corresponding sequence data in GenBank.

Total genomic DNA of these two “stealth” MRSA isolates and their revertants were extracted using the TaKaRa MiniBEST Bacteria GenomicDNA Extraction Kit (TaKaRa Bio Inc., Beijing, China). The whole genomes of wild-type “stealth” MRSA isolates and one revertant of the nasal swab strain were sequenced at the Beijing Genomics Institute using the long-reads PacBio third-generation sequencing method (Pacific Biosciences of California, Inc., Menlo Park, CA, USA) and the short-reads Illumina NovaSeq PE150 platform (Illumina, San Diego, CA, USA). After filtering low-quality reads, the clean data were obtained, which were preliminarily assembled using Link v5.0.1 and subsequently corrected with the Illumina data. Then, Rapid Annotation using the Subsystem Technology v2.0 server was used for gene annotation. Using BLAST, comparative genome analysis was performed among the parental and variant strains. The mutated genes were then confirmed using Sanger sequencing, and the mecA gene of revertants from the clinical strain was analyzed using conventional sequencing, as previously described by us (1 (link)). The sequences were aligned and further analyzed using the Qiagen CLC Genomics Workbench (33 ).

The genomic DNA of strain PR1 was isolated and purified by using a TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit (TaKaRa, Dalian, China). The genomic DNA GC content was determined by HPLC according to the method of Mesbah et al. [14 (link)], with Escherichia coli K-12 as a reference. Cellular fatty acids were extracted according to the MIDI protocol [15 (link)] and identified by using the standard MIDI Sherlock Microbial Identification System (version 6.0).