Easypure genomic dna extraction kit

Total RNA from A. cerana cerana was extracted and cDNA was synthesized using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and an EasyScript First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China), respectively, as per the manufacturers’ protocol. For expression profile analysis of AccDpp at different development and under different types of abiotic stresses, whole honeybee was used to extract RNA, and for the analysis of the expression patterns of AccDpp at different development, the RNA was extracted from different tissues. The extraction of genomic DNA was performed according to the instructions offered by the EasyPure Genomic DNA Extraction Kit (TransGen Biotech, Beijing, China).

E. coli genomic DNA was extracted using Easypure genomic DNA Extraction kit (TransGen Biotech, Beijing, China), and MTB H37Rv genomic DNA was extracted according to previously described protocols [18 (link)]. The primers were designed using Primer 5.0 software for the amplification of lysC and asadh gene based on the sequences published in the NCBI database (GenBank accession number: 948531 and 885118). The primer sequences for amplifying lysC gene (1350 bp) were 5′-TTTTGAATTCATGTCTGAAATTGTTGTCT-3′ (EcoRI, sense) and 5′-AAAAAAGCTTTTACTCAAACAAATTACTA-3′ (HindIII, anti-sense). The primer sequences for amplifying asadh gene (1038 bp) were 5′-TTTTGAATTCATGGGCCTGTCAATAGGGATC-3′ (EcoRI, sense) and 5′ AAAAAAGCTTTCACAAGTCGGCGGTCAGC-3′ (HindIII, anti-sense). The PCR products were digested with EcoRI and HindIII and cloned into the corresponding sites of pET28a(+) respectively. The ligation mixtures were transformed into E. coli DH5α competent cells respectively, and sub clones were sequenced to confirm no mutations in these fragments. The correct plasmids were designated as pET28a(+)-LysC and pET28a(+)-ASADH.

Before performing DNA extraction, clinical samples were washed twice with normal saline by centrifugation to remove ethanol. Genomic DNA was extracted from pellets using the EasyPure Genomic DNA Extraction Kit (TransGen Biotech Ltd., Beijing, China), according to the instructions recommended by the manufacturer. DNA concentration and purity was determined by the ratio of optical density at 260 and 280 nm in a NanoDrop spectrophotometer (Thermo Scientific), and samples were then stored at −20 °C until used in PCR amplification.