Genomic mini ax bacteria spin kit

The DNA isolation from juice samples (3 mL of each sample) was performed using the Genomic Mini AX Bacteria Spin Kit (A&A Biotechnology, Gdynia, Poland). The procedure was carried out according to the instructions provided and recommended by the manufacturer. Isolate concentration was assessed by a fluorimetry method on the Qubit 3.0 device (ThermoFisher Scientific, Waltham, MA, USA) with the use of the dsDNA HS Assay Kit (ThermoFisher Scientific). For each of the samples, three DNA extractions were performed and later combined after a positive quantification.

Bacterial genomic DNA was extracted using a Genomic Mini AX Bacteria Spin Kit (A&A Biotechnology). 16S rDNA was amplified using primers 16SP1 and 16SP2 (Tailliez et al. 2006 (link)). The housekeeping genes coding the glutamyl-tRNA synthetase catalytic subunit (gltX), recombinase A (recA), DNA polymerase III beta chain (dnaN), and subunit B of DNA gyrase (gyrB) were amplified as described earlier (Kazimierczak et al. 2016 (link)). The PCR products were ligated into plasmids pJET1.2 (Thermo Fisher Scientific) according to the manufacturer’s instructions. The plasmids were transformed in E. coli XL1 Blue using a standard method. The inserts of positive clones were sequenced from both strands in Genomed (Poland).

The strains were cultured in LB liquid medium overnight at 37 °C with shaking at 180 rpm. Genomic bacterial DNA was extracted using the Genomic Mini AX Bacteria Spin kit (A&A Biotechnology, Gdynia, Poland), according to the manufacturer’s instruction. The samples were stored at 20 °C.

Isolation of genomic DNA was performed using the Genomic Mini AX Bacteria Spin Kit (A&A Biotechnology), according to the manufacturer’s procedure.

DNA isolation from milk samples was performed using the Genomic Mini AX Bacteria Spin kit (060-100S, A&A Biotechnology, Gdańsk, Poland) according to the protocol provided by the manufacturer. Finally, the purified DNA was eluted. The isolates were stored at −80 °C after they had been neutralized, in order to minimize matrix degradation.
The efficiency of isolation was checked each time based on the fluorimetric method with the use of the Qbit 3.0 device and the Qubit™ dsDNA HS Assay Kit (Q32851, ThermoFisher Scientific, Waltham, MA, USA). For each sample, three DNA extractions were performed and finally combined after a positive quantification.

Total genomic DNA was extracted while using a Genomic Mini AX Bacteria Spin Kit (A&A Biotechnology, Gdynia, Poland and then stored at −20 °C. All of the PCR amplifications were carried out with PCR mix RAPID (A&A Biotechnology, Gdynia, Poland), according to the manufacturer’s recommendations. The primer sequences and PCR conditions used are listed in Table 3. The amplified PCR products were purified with Clean-Up purification columns (A&A Biotechnology, Gdynia, Poland) and then sequenced in Genomed S.A. (Warsaw, Poland). Preliminarily, the 16S rRNA gene sequences were compared with the EzBioCloude database. All of the obtained sequences were analyzed while using BLAST available on the NCBI website. Multiple sequence alignment matrices of the individual gene sequences were created using ClustalW included in the MEGA 6.06 software [56 (link)]. The sequence identity values were calculated while using BioEdit 7.0.5 software.
The 16S rRNA gene sequence similarity threshold value of 98.7% between the isolate and species type strain was used as an indicator that an isolate can be a member of a given species [34 ]. The identification of the isolates at the species level was considered to be final when the searching results that were based on 16S rRNA gene sequences were concordant with those based on the rpoB, rpoD, or recA gene sequences.

In order to isolate L. monocytogenes genomic DNA, the column method with the Genomic Mini AX Bacteria Spin Kit (A&A Biotechnology, Gdynia, Poland) was applied, according to the protocol provided by the manufacturer.