The isolates were further serotyped by the molecular method published by Howell et al., 2015, following modifications by Schuwerk et al., 2020 [12 (link)], which divided the assay into two sets of multiplex PCR (mPCR). The PCR reactions were set up consisting of 12.5 µL 2× Mytaq™ HS Red Mix PCR master mix (Meridian Bioscience®, Cincinnati, OH, USA), 0.5 nM of forward and reverse primer each, 2 µL (50–100 ng/µL) template and nuclease-free water topped-up to a volume of 25 µL. The PCR assay was performed at 95 °C for 1 min, followed by 30 cycles of 95 °C for 15 s, 58 °C for 30 s and 72 °C for 30 s, and a final extension of 72 °C for 5 min. All PCR products were subjected to 2% agarose gel electrophoresis in TAE buffer 80 V for 45 min, using a 100-bp molecular weight marker (Qiagen, Hilden, North Rhine-Westphalia, Germany) as a guide. Electrophoresed gels were visualized using a UV transilluminator and gel documentation system (Syngene, Frederick, MD, USA). The PCR bands were measured using the Image Lab software v6.1 (Bio-rad Laboratories, Inc., Hercules, CA, USA).
Uv transilluminator and gel documentation system
Manufactured by Syngene
Sourced in United States
A UV transilluminator is a device that emits ultraviolet light to visualize and analyze DNA or protein samples after they have been separated by gel electrophoresis. The gel documentation system captures digital images of the illuminated gel, allowing for further analysis and documentation of the results.
Automatically generated - may contain errors
2 protocols using uv transilluminator and gel documentation system
1
Multiplex PCR Serotyping of Isolates
2
Virulence Typing of Glaesserella parasuis
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One forward and two reverse primers, outlined by Galofré-Milà et al., 2017 [11 (link)], were used to distinguish the vtaA gene of G. parasuis into virulent and nonvirulent strains. The PCR reactions were set up comprising of 12.5 µL 2× Mytaq™ HS Red Mix PCR master mix (Meridian Bioscience®, Cincinnati, OH, USA), 0.5 nM of forward and reverse primer each, 2 µL (50–100 ng/µL) template and nuclease-free water topped-up to a volume of 25 µL. The PCR assay was performed at 95 °C for 1 min, followed by 30 cycles of 95 °C for 15 s, 54 °C for 30 s, 72 °C for 30 s and a final extension of 72 °C for 5 min. The PCR products were subjected to 2% agarose gel electrophoresis in TAE buffer 80 V for 45 min, using a 100-bp molecular weight marker (Qiagen, Hilden, North Rhine-Westphalia, Germany) as a guide. Electrophoresed gels were visualized using a UV transilluminator and gel documentation system (Syngene, Frederick, MD, USA). The PCR bands were measured using the Image Lab software v6.1 (Bio-rad Laboratories, Inc., Hercules, CA, USA).
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