T3 super pcr mix

Agarose gel electrophoresis procedure was conducted as previously described 21 (link), 22 (link). Firstly, we performed PCR enrichment of the divergent primers of 4 circular RNAs in both cDNA and gDNA by using 1.1 × T3 Super PCR Mix (TsingKe, Nanjing, China). PCR conditions included: pre-denaturation (98˚C, 30 sec, 1 cycle) and PCR reaction (98˚C, 15 sec, 60˚C, 10 sec, 72˚C, 10 sec, 35 cycles in total) followed by 72˚C for 7 min in 1 cycle. Then, the PCR products were checked on 1% agarose gel electrophoresis. The results are presented in Supplemental Figure S1.

Genomic DNA was extracted from dried apothecia using the CTAB procedure with some modification (Doyle and Doyle, 1990 ). The large subunit of the nuclear ribosomal RNA (Partial LSU), the translation elongation factor 1-alpha (TEF), and the heat shock protein 90 (HSP90) were amplified by polymerase chain reaction (PCR) using universal and/or previously published primers LR0R/LR5 (Vilgalys and Hester, 1990 (link)) and H_LSUf1/H_LSUr2 (Landeros et al., 2015 (link)), EF595F/EF1160R (Skrede et al., 2017 (link)), and H_hspf and H_hspr (Skrede et al., 2017 (link)) (Table 1). PCR amplifications were performed in a total volume of 25 μl, containing 21 μl 1.1 × T3 Super PCR Mix (Tsingke TSE030, Tsingke Biological Technology Co.), 1 μl of each primer, and 2 μl of DNA template. PCR reactions were carried out in an Applied Biosystems 2720 Thermal Cycler (Foster City, CA, USA) under the following conditions: an initial denaturation at 98°C for 5 min, followed by 34 cycles of denaturation at 98°C for 25 s (30 s for LSU and HSP90), annealing at 53°C for 30 s (52°C for LSU: H_LSUf1/H_LSUr2, 58°C for HSP90), and extension 45 s at 72°C, followed by a final extension at 72°C for 7 min. PCR products were verified by electrophoresis with 1% ethidium bromide-stained agarose gel. Those presenting the target genes have been sent to Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China, for sequencing.

Genomic DNA of 100 gilts (50 Large White and 50 Mi gilts) was extracted from ear tissue by a standard phenol/chloroform method. The specific primers (Supplementary Table S1) were designed using Primer5 to amplify 5’-UTR, 5 exons and 3’-UTR of the porcine HSD17B14 gene. PCR reactions were performed using 1.1 × T3 Super PCR Mix (TsingKe, Nanjing, China). The amplified PCR products were sequenced, and multiple sequence splices and alignments were performed to analyze the SNPs in Large White and Mi gilts using the software DNAMAN 8.0 (https://www.lynnon.com/index.html) and Chromas (v 2.6.4, Technelysium Pty Ltd., South Brisbane, Australia).

DNA was extracted using the CTAB method [31 (link)]. ITS region of ribosomal DNA and the D1/D2 domains of the ribosome subunit (LSU) were amplified and sequenced with the primer pairs of ITS1/ITS4 (ITS1 5′ —GTC GTA ACA AGG TTT CCG TAG GTG— 3′; ITS4 5′ —TCC TCC GCT TAT TGA TAT GC— 3′) and NL1/NL4 (NL1 5′ —GCA TAT CAA TAA GCG GAG GAA AAG— 3′; NL4 5′ —GGT CCG TGT TTC AAG ACG G— 3′) [32 ,33 (link)].
The PCR reaction was performed in the 25 µL reaction mixture containing 0.5 µL of each primer (10 pM/µL), 1.0 µL of genomic DNA (10 ng/µL), and 23 µL of 1 × PCR Master Mix buffer (T3 Super PCR Mix, 10 × 1.125 mL, Tsingke Biotechnology Co., Ltd., Beijing, China). Amplification was performed in an AB 2720 thermal cycler (Applied Biosystems, Foster City, California, USA), with the program consisting of 98 °C for 2 min, 35 cycles of 98 °C for 10 s, 52 °C for 10 s, and 72 °C for 15 s, and the last elongation at 72 °C for 5 min.

All the collected samples were detected for PPV7, PCV2, PCV3, and PCV4 using primers listed in Table 1. The primers for PCV2, PCV3, and PCV4 detection were all synthesized according to previous studies (21 (link), 22 (link)). The PPV7-2F/2R were designed for PPV7 detection and genome amplification. In this study, 25 μl PCR (Polymerase chain reaction) system was applied, containing 22 μl T3 Super PCR Mix (Tsingke Biotechnology, Beijing, China), 1 μl of 10 μM each of the two primers, and 1 μl genomic DNA. The following PCR reaction conditions were used: pre-denaturation at 98°C for 2 min, 35 cycles of denaturation at 98°C for 10 s, annealing at 58°C for 15 s, extension at 72°C for the 20 s, and a final extension at 72°C for 5 min. Finally, the PCR products were analyzed by 1.5% agarose gel electrophoresis.