Gel doc uv transilluminator system

Frozen tissue samples of cases 5, 6 and 7 were submitted to PCR for the detection of Brucella spp. by hemi-nested PCR targeting an outer membrane protein gene of B. abortus [54 (link)].
The reactions were loaded as previously reported using B. suis bv 2 strain Thomsen as positive control and no template control as negative control.
Specifically, molecular detection was attempted on CNS, spleen, lung, liver, pre-scapular, tracheobronchial, pulmonary lymph nodes, tongue and skin ulcers, laryngeal tonsil and CSF of case 5; on CNS of case 6; on CNS, spleen, liver, lung and testes of case 7. For DNA extraction, tissue samples (30–50 mg) were physically disrupted using a TissueLyser II homogenizer (Qiagen, Hilden, Germany) by high-speed shaking in plastic tubes with stainless steel beads (5 mm in diameter). Genomic DNA was then extracted from the disrupted tissues with an AllPrep DNA/RNA Mini kit (Qiagen) according to the manufacturer’s instructions.
The PCR products were analyzed by electrophoresis on 2% agarose gel containing GelRed (Biotium, Fremont, California, USA), compared with molecular weight markers and subsequently photographed on a Gel-Doc UV transilluminator system (Bio-Rad, Hercules, California, USA).

The DNA was isolated from the Blastocystis culture, which was centrifuged in 400× g 5 min. The genetic material was extracted from the sediment using a commercial Genomic Mini kit (A&A Biotechnology) in accordance with the manufacturer’s instructions. The samples were stored at –20 °C for future usage.
PCR was carried out following the method described by Grabensteiner and Hess [22 (link)] at the following cycling conditions: initial denaturation at 94 °C for 15 min followed by 40 cycles of 94 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min, and a final extension at 72 °C for 10 min. The amplification products were resolved in a 2% agarose gel, stained with Midori Green, and visualized using Gel-Doc UV transilluminator system with Quantity One software (Biorad). The products (size ~500 bp) from 5 cultures were sequenced to confirm the genus Blastocystis belonging. The obtained sequences were analyzed using Mega X and compared with GenBank sequences. One sample from the parasite culture was chosen as a positive control for future investigation, and the sequence obtained was placed in the GenBank database under accession number MZ956599.