Digoxigenin-Labeled Probe Synthesis and Southern Blotting

Primers in Table 1 were designed to amplify the Digoxigenin (DIG)-labeled probe with a PCR DIG Probe Synthesis Kit (Roche, Basel, Switzerland). Approximately 20 μg of gDNA was obtained from CFN and digested with restriction enzymes (Dra I and EcoR I) (Figure S4). The digested DNA products were separated by 0.8% (w/v) agarose gel electrophoresis and transferred to a Hybond N⁺ membrane (Amersham-Biosciences, Little Chalfont Buckinghamshire, England) [27 (link),40 (link)]. The membrane was hybridized for 18 h at 42 °C with the probe. Hybridization was performed using a Dig High Primer DNA Labeling and Detection Starter Kit I (Roche, Basel, Switzerland) according to the manufacturer’s instructions. After hybridization, the membrane was washed with 2 × SSC/0.1% SDS for 15 min at 25 °C followed by 0.5 × SSC/0.1% SDS for 30 min at 65 °C and examined. Equal amounts of carrot callus gDNA and plasmid pET-32a-Ar-far-1 were used as a control group [39 (link)].