Dna extraction kit

Plant genomic DNA preparation and polymerase chain reaction (PCR) analysis were conducted as described in Song et al. (2015) (link). Genomic DNA was isolated from the rosette leaves of Arabidopsis plants using a DNA extraction kit (RBC Bioscience, Seoul, Korea), according to the manufacturer’s recommendations. PCR analysis was performed to confirm the presence of HC (281 bp) and LC (227 bp) genes of T₁ plants. Primer sets were described as follows: HC forward primer, 5′-CAG ACT CAC CAT TAC CGC-3′; HC reverse primer, 5′-AGT AGT CCT TGA CCA GGC-3′; LC forward primer, 5′-CAC TGG AAC CAG CAG TGA-3′; LC reverse primer, 5′-TGT AGT CGC CTG CAT ATG A-3′. The PCR reaction was subjected to 26 cycles of 94 °C for 20 s, 58 °C for 10 s, and 72 °C for 30 s. The PCR products were analyzed by electrophoresis in a 1.0% agarose gel with ethidium bromide, and visualized under UV illumination. The pBI mAb 57 vector was used as a positive control, and genomic DNA extracted from Col-0 Arabidopsis was used as a negative control.

Rosette leaves (approximately 100 mg) from four-week-old NT and transgenic plants expressing mAb CO were used for polymerase chain reaction (PCR) analysis. A DNA extraction kit (RBC Bioscience, Seoul, Korea) was used to extract genomic DNA from plant leaves, following the manufacturer’s recommended protocol. PCR was performed to confirm the presence HC (1,416 bp) and LC (717 bp) genes associated with mAb CO, and transformants were selected from T₁ plants. Primers were designed as follows: HC forward primer, 5′-GCGAATTCATGGAATGGAGCAGAGTCTT TATC-3′; HC reverse primer, 5′-GATTAATCGATTTTACCCGGAGTCCG-3′; LC forward primer, 5′-GCCTCGAGATGGGCATCAAGATGGAATCACAG-3′; LC reverse primer, 5′-GAGGTACCCTAACACTCATTCCTGTTGAAGCTC-3′. Leaves from NT plants were used as a negative control, and the pBI CO gene was used as a positive control. PCR analysis was replicated more than three times.

A total of 266 adult P. leucomystax complex individuals were collected from 15 different localities in Thailand (Table 1). All samples were dissected to obtain the liver, which was then stored in absolute ethanol. Sample collection and euthanization were approved by the Center For Animal Research Naresuan University under project number NU-AE591028. Genomic DNA was extracted from liver tissue using a DNA extraction kit (RBC Bioscience, Singapore) and kept at −20 °C for further use. Individual DNA was used as a template for PCR amplification of the mitochondrial COI gene using Taq DNA polymerase in a total volume of 25 µL under the following conditions: an initial denaturation at 94 °C for 5 min, followed by 35–40 cycles at 94 °C for 30 s, 50 °C for 30 s and 72 °C for 1 min, and a final extension step at 72 °C for 7 min. PCR products were visualized on 1.5% agarose gel under UV illuminator. The expected size of the partial mitochondrial COI gene sequence was 688 bp. Subsequently, all PCR products were purified using a QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) and then sequenced (Macrogen, Seoul, South Korea).