Pcr purification kit

Genomic DNA was extracted by using a phenol-chloroform extraction method [35 (link)]. The bacterial universal primers (final concentration of 0.4 μM) 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-CGGTTACCTTGTTACGACTT-3′) [36 (link)], together with the AppTaq RedMix (Appleton, Birmingham, UK) reaction mixture, were used to amplify the 16S rRNA gene. The PCR was performed with an initial denaturation step at 95 °C for 3 min, 30 cycles of denaturation at 95 °C for 15 s, annealing at 55 °C for 15 s, extension at 72 °C for 30 s, and a 5-min final extension at 72 °C. The PCR products were purified by using a PCR purification kit (Sigma; GenElute, St Louis, MO, USA). Sanger sequencing was performed by Eurofins Genomics (Ebersberg, Germany). Chromas software (version 2.6.6; Technelysium, South Brisbane, Australia) was used to trim low-quality sequences and Bioedit (version 7.05.3) [37 (link)] was used to merge forward and reverse DNA sequences. The 16S rRNA sequence was subjected to BLASTn to identify the closest relatives. An isolate (13f), which was identified as Alcaligenes, was selected for further study. The sequence was deposited into the NCBI database (accession number MZ323998).

Chromosomal DNA from the pure cultures of the emetic food poisoning isolates was extracted using the CTAB/phenol-chloroform extraction method described by Griffiths et al. (2000) [59 (link)]. The 16S rRNA gene was amplified using primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-CGGTTACCTTGTTACGACTT-3′) [60 (link)] at a final concentration of 0.1 μM. The PCR was performed with an initial denaturation for 2 min at 94 °C, 35 cycles of denaturation for 30 s at 94 °C, primer annealing for 30 s at 55 °C, extension for 30 s at 72 °C, and a final extension for 7 min at 72 °C (Lane, 1991) [61 (link)]. The PCR products were purified using a PCR purification kit (Sigma-Aldrich, Darmstadt, Germany). Sequencing was performed by Celemics (Seoul, Republic of Korea). The low-quality bases in the sequences were trimmed using Bioedit software version 7.05.3 [62 (link)]. The processed sequences were subjected to Basic Local Alignment Search Tool (BLAST) analysis against the 16S rRNA sequences in the National Center for Biotechnology Information (NCBI) database to find the closest relatives.

PCR reaction was performed in total volume of 25 μl, using Thermocycler (Germany). The reaction mixture consisted of Taq DNA Polymerase 2× Master Mix RED with 1.5 mM MgCl₂ (Amplicon, Denmark), specific primers and DNase-free water. Amplification was carried out at 95 °C for 30 s in order to pre-denature the template DNA, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 1 min and extension at 72 °C for 1 min. The final extension was completed at 72 °C for 5 min. The PCR products were purified using PCR purification kit (Millipore), and subjected to sequencing (First Base Co, Malaysia) using the BigDye® Terminator v3.1 cycle sequencing kit chemistry. Then they were loaded into DNA Analyzer (GATC Biotech). Both sense and antisense strands of PCR products were directly sequenced using the same primers as the PCR amplification. SNPs were detected by direct sequence analysis using DNAsis MAX.3 software. The reference sequence for the IFITM3 gene was based on the sequence of human chromosome 11, clone GRCh38.p2.

The PCR products were purified using Microcon (Millipore) PCR purification kit according to the manufacture’s procedure, and the concentration of DNA was measured by nano-drop spectrophotometer. Each sample was sequenced by both forward and reverse primers. After ethanol precipitation, the purified PCR products were subjected to sequencing in the ABI prism 3130 Genetic analyzer (ABI prism, USA).