Gel doctm xr with image labtm software

The modified CTAB (Cetyltrimethylammonium bromide) method was used for DNA extraction from bacteria [25 ]. The DNA was extracted from purified (on Tryptic Soya Broth (TSB) for 24 h at 37 °C) and biochemically confirmed Staphylococcus aureus. Briefly, 10% Sodium Dodecyl Sulfate (SDS), Proteinase K (Thermo Scientific™ Proteinase K), 10% CTAB/Sodium chloride (NaCl), and 5M NaCl were used for cell lysis. Extracted DNA was suspended in 100 µL of Tris-Ethylenediaminetetraacetic acid buffer (TE Buffer) and stored at −20 °C. Qualitative measurement of DNA was performed using a 0.5% agarose gel. The Gel Doc system (BIO-RAD Gel Doc^TM XR + with Image Lab^tm Software, BioRad, Hercules, CA, USA) was used for gel visualization.

The RNA (OMEGA, R6934-01, USA) was isolated from THJ-16T and THJ-21T cells without and with resveratrol treatment for 48 h. Nanodrop (Thermo Scientific) was used to evaluate the quality and concentration of RNA extracted. Reverse transcription was performed using Takara PrimeScript^TM RT reagent kit (Takara Biotechnology Co., Ltd., RR037A, Japan). Briefly, 0.5 μg of each RNA sample was added to 10 μl of RT reaction mixture containing 2 μl of 5× PrimeScript buffer, 0.5 μl of PrimeScript RT Enzyme Mix Ⅰ, 0.5 μl of oligo dT-adaptor primer, 0.5 μl of random 6 mers and RNase-free distilled H₂O up to 10 μl. The reaction was carried out at 37°C for 15 min, at 85°C for 5 sec. The synthesized cDNA was used as a template for the PCR reaction using Premix Taq^TM (Takara Biotechnology Co., Ltd., RR902A, Japan) and primers for target genes are listed in Table 1. Furthermore, RT-PCR was carried out by Bio-Rad T100 thermal cycler (Bio-Rad, Richmond, CA). The PCR condition was performed as follows: the samples were subjected to 30 cycles at 98°C for 10 s, 55°C for 30 s, 72°C for 60 s, the samples were stored at 4°C. Agarose gels (1.0%) containing ethidium bromide (0.5 mg/mL) were prepared for the separation of the PCR products, and BIO-RAD Gel Doc^TM XR⁺ with Image Lab^TM Software (BIO-RAD, USA) was used to visualize and photograph the samples. mRNA levels were normalized to levels of GAPDH.

Antibiotic resistance genes (mecA and mecC) were amplified by using a basic Thermal Cycler PCR (BIO-RAD T100^TM Thermal Cycler, Massachusetts, USA) in which specific primers were used for both mecA and mecC genes (Table 1). A total volume of the PCR reaction (15 µL) contained 2 µL (10 ng/µL) of DNA, 7.5 µL of 2X Taq master mix (Vazyme Biotech Co., Nanjing, China), 1 µL (10 µM) of forward primer, 1 µL (10 µM) of reverse primer, and 3.5 µL of deionized water. PCR for both genes (mecA and mecC genes) was performed by applying the following conditions: first denaturation at 95 °C for 5 min followed by 35 cycles of 95 °C for 1 min, 55 °C for 30 s, 72 °C for 1 min, and 72 °C for 5 min [21 (link),22 (link)]. Molecular detection of mecA and mecC was performed by loading 6–7 µL of 100 bp DNA ladder into the first well and the remaining wells were loaded by PCR products using 1.5% agarose gel containing 0.5 mg/mL of ethidium bromide in Tris-borate-EDTA (10 mM+1mM EDTA; pH 8.0) buffer used by applying 120 V for an hour. DNA bands were observed under ultraviolet light (UV-light) using the Gel Doc system (BIO-RAD Gel Doc^TM XR + with Image Lab^tm Software, BioRad, Hercules, CA, USA).