Dna extraction kit

A qPCR-based method was used for mitochondrial DNA content detection. Briefly, total DNA was extracted from HCC cells with a DNA Extraction Kit (ThermoFisher) according to the manufacturer’s guidelines. Then, extracted total DNA was used for real-time PCR analysis for the levels of mitochondrial ND1 and nuclear HGB. Relative mitochondrial DNA content was calculated by the ratio of the mitochondrial ND1 gene to nuclear HGB. The primer sequences for ND1 and HGB are provided in Table S1.

The genomic DNA from each isolate was extracted using the DNA extraction Kit (Thermo Fisher), following the manufacturer’s instruction. The sequences of intercellular adhesion (ica) genes icaA and icaD (accession number U43366) were taken from the GenBank sequence of the National Center for Biotechnology Information (NCBI) database. Primers specific for icaA and icaD were designed by the Primer3 program and were purchased from Solis Biodyne, (Denmark). The primer used for the detection of icaA was forward 5’-TCTCTTGCAGGAGCAATCAA-3’ and reverse 5’-TCAGGCACTAACATCCAGCA-3’ primer. The two primers include a 188-bp region. For detection of icaD, 5’- ATGGTCAAGCCCAGACAGAG-3’ was used as a forward primer and 5’-CGTGTTTTCAACATTTAATGCAA-3’ was used as a reverse primer with the product size of 198 bp [30 (link)].

Identified P. aeruginosa isolates were sub‐cultured on TSB media (Merck, Germany) and incubated for 48 h at 37°C in an aerobic condition. According to the manufacturer's instructions, the genomic DNA was extracted from the isolates using the DNA extraction kit (Thermo Fisher Scientific, St. Leon‐Rot, Germany). The purity (A260/A280) and concentration of the extracted DNA were then checked (NanoDrop; Thermo Scientific, Waltham, MA, USA). Furthermore, the DNA's quality was assessed on a 2% agarose gel stained with ethidium bromide (0.5 µg/ml) (Thermo Fisher Scientific, St. Leon‐Rot, Germany) (Dehkordi et al., 2020 (link)).

Distinctive colonies of H. pylori were additionally approved using the 16S rRNA-based PCR method. Typical colonies were sub-cultured on Wilkins Chalgren anaerobe broth supplemented with same materials mentioned above [15 (link)]. Genomic DNA was then extracted from colonies using a DNA extraction kit (Thermo Fisher Scientific, St. Leon-Rot, Germany). Procedure was performed rendering to the manufacturer’s guidelines. Purity (A260/A280) and concentration of extracted DNA were then checked (NanoDrop, Thermo Scientific, Waltham, MA, USA). The truth of the DNA was assessed on a 2% agarose gel stained with ethidium bromide (0.5 μg/mL) (Thermo Fisher Scientific, St. Leon-Rot, Germany). Polymerase Chain Reaction (PCR) was performed using a PCR thermal cycler (Eppendorf Co., Hamburg, Germany) according to reported procedure [15 (link)].

Genomic DNA from Xenophilus sp. SN213 and Stenotrophomonas sp AS was isolated using a DNA extraction kit (Thermo Scientific), partially digested by Sau3AI, and fragments in the range of 2–4 kb were isolated by gel electrophoresis. The resulting fragments were cloned into BamH1-digested pET28a, (pET28a has been used successfully in our laboratory for cloning and overexpression of many genes) and then transformed into competent cells of E. coli BL21 (DE3) RIL. The transformants were screened on LB-Kan-folate-IPTG plates (containing 32 μg/mL kanamycin, 0.5% (v/v) folate and 0.1% IPTG). Positive clones of yellow color were picked and plasmids isolated as described above.

Total genomic DNA from E. coli was isolated using a DNA extraction kit (Thermo Fisher Scientific), according to the manufacturer’s instruction. Gel retardation experiments were performed by mixing 150 ng of the DNA and increasing amounts of p4 in buffer containing 10 mM Tris–HCl, 1 mM EDTA, pH 8.0. The mixtures were incubated at RT for 30 min, and subsequently analyzed by electrophoresis on a 0.8% agarose gel in the TBE buffer (90 mM Tris-borate, 2 mM EDTA, pH 8.3).