Ez 10spin column gel extraction kit

Primers 3CS and 5CS were used to amplify the variable regions of the class 1 integron-positive isolates. LA Taq DNA polymerase (TaKaRa Biotechnology, Japan) was used for the PCRs. After electrophoresis on a 1% agarose gel, the PCR products were excised and purified using the EZ-10Spin Column Gel Extraction Kit (Bio Basic). All of the PCR products were sequenced by primer walking, starting with the 3CS and 5CS primers (Table 1). Primer design and DNA sequencing were performed by Sangon Biotechnology Co., Ltd. (Shanghai, China). Nucleotide sequences were analyzed and compared using BLAST software (National Center for Biotechnology Information; www.ncbi.nlm.nih.gov).

Because boiling method is a rough extraction method, which nucleic acid concentration may decrease over time, this method can be used in the preliminary screening of integron. To ensure the reproducibility and stability of the experimental results, the reagent extraction method should be used in further experiments. Therefore, we adopt the method of extracting DNA by kit to screen the variable region of integron. Total genomic DNA was isolated from the class 1 integron-positive strains using the EZ-10 Spin Column Bacterial Genomic DNA Miniprep Kit (Bio Basic, Canada) according to the supplier's instructions. Table 1 shows the primers used in this study. Primers 5CS and 3CS were used to amplify the variable regions of the class 1 integrons.
For the PCRs, LA Taq DNA polymerase (TaKaRa Biotechnology, Japan) was used according to the manufacturer's instructions. After electrophoresis on a 1% agarose gel, the PCR products were excised and purified according to the manufacturer's instructions of the EZ-10 Spin Column Gel Extraction Kit (Bio Basic). All amplicons were sequenced by primer walking, starting with 3CS and 5CS. Nucleotide sequences were analyzed and compared using BLAST software.