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This study evaluates the PdKn1 gene copy number using Southern blot analysis, employing the internal sequence of the gene as a probe. The analysis incorporates Eco RV and Hind III restriction enzymes to digest genomic DNA samples. The digested DNA was subjected to electrophoresis on a 0.8% agarose gel and subsequently transferred onto a Hybond+ nylon membrane. Hybridization was conducted overnight at 61°C, with stringent washes following. The findings reveal at least five distinct bands upon digestion with Hind III, indicating gene copy variations.
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Kb 10 5 3 1.5 1 Figure 1 - Supplementary Hind III Eco RV Figure 1 –PdKn1 gene copy number. Southern blot analysis for PdKn1 using as probe the internal sequence of the gene (amino acid region b-d in Figure 2A) that contains two very conserved motives in Knox genes. Eco RV cuts once inside the probe template. For PdKn1 Southern blot analysis Eco RV and Hind III restriction enzymes were used separately to digest 10mg aliquots of genomic DNA. Digested DNA was electrophoresed in a 0.8% 1xTAE agarose/EtBr gel, run overnight at 2V/cm and blotted overnight onto a Hybond+ nylon membrane. The hybridisation was performed overnight at 61C, and the stringency washes, the first (1xSSC and 0.1% SDS) and second (0.5xSSC and 0.1% SDS), were run at 61C for 45 and 20 min, respectively. The membrane was exposed to a Kodak film for 1h at room temperature, in the dark. Digestion with Hind III reveals at least 5 bands
