Fastgene fas digi pro

The PCR reactions were performed to detect the DNA of bacteriophages vB_SenM-2 and vB_Sen-TO17 in the plasma of chickens. For this purpose, 10 µl of Color Taq PCR Master Mix (2×), 2 µl of specific forward and reverse primers each (final concentration of 1 µM), 5 µl of nuclease-free water, and 1 µl of matrix DNA (concentration 10 ng/µl) were mixed. The reaction was conducted in Mastercycler® nexus (Eppendorf, Germany) with the following parameters: number of cycles—30; denaturation—15 s, 94 °C; annealing—15 s, 55 °C; extension—60 s, 72 °C. Then, the samples were loaded into wells of 1.5% agarose gel (agarose solution in Tris-octane-EDTA buffer (Bioshop, Canada) supplemented with SimplySafe^TM (EURx, #E4600-02). Electrophoresis was set at 100 V for 30 min. DNA was visualized by UV light using a gel documentation system (FastGene FAS-DIGI PRO, Nippon Genetics Europe, Germany) with the following parameters: aperture 5.6 AV, exposure 1/40 TV, ISO 800.

The obtained PCR reaction products were visualized in a 1.5% agarose gel (agarose solution in Tris-Octane-EDTA buffer (Bioshop, Canada) supplemented with SimplySafe™ (EURx, Poland) solution according to the manufacturer’s instructions). Electrophoresis was conducted for 30 minutes at 100 V. The gels were then visualized using a gel documentation system (FastGene FAS-DIGI PRO, Nippon Genetics Europe, Germany). Parameters of the images taken: aperture 9 AV, exposure 1/50 TV, ISO 1600.

RNA extraction was performed using TRIzol reagent (Invitrogen, Waltham, MA, USA), followed by quantification using a NanoDrop 2000 spectrophotometer (Thermo Scientific) and integrity assessment with FastGene FAS-DIGI PRO (NIPPON GENETICS EUROPE GmbH, Düren, Germany). Reverse transcription utilized the PrimeScriptTM RT Reagent Kit (Takara, Shiga, Japan) to generate cDNA. Quantitative PCR was conducted on the BIO-RAD CFX96 Real-Time PCR Detection System using TOPreal™ SYBR Green qPCR UDG PreMIX (Enzynomics, Munich, Germany) with cycling conditions of 95 °C for 5 min, followed by 40 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s. Melt curve analysis was performed at 55 °C for 2 s. Data were analyzed using GraphPad Prism 9 software (La Jolla, CA, USA), presenting results as fold changes or relative expression levels compared to a reference or control group.