Bacterial genomic dna extraction kit

Enterobacterial repetitive intergenic consensus (ERIC) polymerase chain reaction (PCR) analysis was used to analyze the microbial community structure of rats (14). DNA was extracted from each fecal sample with a bacterial genomic DNA extraction kit (QIAgen, Germany). PCR amplification was performed on extracted DNA as described by Khosravi et al. [22 (link)] using the primers ERIC1 (5’-ATGTAAGCTCCTGGGGATTCAC-3’) and ERIC2 (5’-AAGTAAGTACTGGGGTGAGCG-3’) in a PCR amplification instrument (Bio-Rad, USA). The reactions were performed in a final volume of 25 μL containing 2 μL of template DNA, 0.5 mL of each ERIC primer, 0.2 μL of 5 U/μL Taq DNA polymerase, 2 μL of 2.5 mmol dNTP, 2.5 μL of 10 × buffer and 17.3 μL nuclease-free water. The amplification parameters were initial denaturation at 94°C for 7 min, 30 cycles of PCR including denaturation at 94°C for 1 min, annealing at 47.3°C for 1 min, extension at 65°C for 8 min and a final extension at 65°C for 16 min. These ERIC-PCR products were separated via electrophoresis in 1% (w/v) agarose gel, stained with 0.5 μg/μL ethidium bromide (Qiagen, Germany) and analyzed under UV light in a gel documentation system (UVP, USA).

In total, 31 LAB strains were used to validate the specificity of the species-specific primers developed in this study (Table 2). Bacterial strains were obtained from the Korean Agricultural Culture Collection (KACC) and from the Belgian Coordinated Collections of Micro-Organisms (BCCM). All strains were routinely cultured at 30 °C for 2 days on de Man Rogosa Sharpe (MRS) agar (Oxoid, Basingstoke, Hampshire, UK). Total genomic DNA was extracted with a bacterial genomic DNA extraction kit from Qiagen (Hilden, Germany). The concentration and quality of the genomic DNA samples were verified using a NanoDrop^® ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA).

All isolates used in this study were maintained on BHI plates with 5% sheep agar under anaerobic conditions. The genomic DNA of these isolates was extracted using a bacterial genomic DNA extraction kit (Qiagen, Germany) according to the manufacturer’s instructions. DNA was then dissolved in 100 μL of TE (10mMTris-HCl1mM EDTA PH = 8.0) and stored at −20°C until use.

The bacterial suspension (300 μL) was extracted using a bacterial genomic DNA extraction kit (Qiagen, Germany) according to the manufacturer’s instructions. The extracted genomic DNA was eluted with 70 μL Tris-EDTA (TE) buffer. DNA samples were quantified using a Qubit fluorescence quantizer (Thermofisher, USA) and stored at −20°C.