Thermal gradient apparatus

Genomic DNA was extracted from 5 ml of sheep blood collected from the jugular vein using rapid salting-out methods.^{27 (link)}
The Primer 3 online program^{28 (link)}
was employed to design PCR primers based on the sequence of the ovine PTX3 (Oar_v4.0; Chr 1, NC_056054.1). To identify the optimal conditions for PCR amplification, we used a Bioneer premix and the Eppendorf thermal gradient apparatus from Germany. The PCR was performed, and thermal gradients were applied to determine the optimal amplification conditions, as detailed in Table 1. The amplification conditions were as follows: 5 minutes of initial denaturation at 94 °C; 30 seconds of denaturation at 94 °C; 45 seconds of annealing; 30 seconds of extension at 72 °C, for a total of 30 cycles; and 5 minutes of final extension at 72 °C followed by storage at 4 °C.^{29 (link)}
Electrophoresis of PCR products on 2% agarose gels was performed and images were captured using a Chemidoc Gel Imager (Bio-Rad, Hercules, California).^{30 (link)}

The genetic analysis of the sheep was conducted using a blood sample collected from its jugular vein. Genomic DNA was extracted from a sample using rapid salting-out methods.^{23 (link)}
Using NCBI Primer-BLAST, all 232 PITX2 genetic sequences were amplified.^{24 (link)}
A polymerase chain reaction (PCR) was performed using a Bioneer premix, and thermal gradients were performed using an Eppendorf thermal gradient apparatus (Germany) to identify the optimal PCR-amplifying conditions (Table 1). The sample was denatured for 4 minutes at 94°C, followed by 30 cycles of 30 seconds at 94°C, then annealed for 45 seconds and elongated for 30 seconds at 72°C. Then, the PCR products were electrophoresed on agarose gels (2%) (Figure 1), and the gel images were visualized with a Chemidoc Gel Imager (Bio-Rad, Hercules, CA, USA).^{25 (link)}