4.4. DNA Isolation and Quantification

The applicable leaflets were halved and dried overnight at 55 °C. The next day, after weighing, the dried leaflets were treated with a lysis buffer consisting of SDS, EDTA, Tris-HCl, saturated sodium chloride, distilled water, and proteinase K for 3 h at 55 °C. The samples were then cooled at 4 °C for 10 min, followed by centrifugation (10 min at 13.2 rpm at 4 °C) to separate the cellular debris and proteins from the DNA. Then, the DNA-containing supernatant was treated with 100% Isopropanol for 15 min at −20 °C. After centrifugation for three minutes at 13.2 rpm at 4 °C, the pellets were washed with 70% Isopropanol and 80% EtOH, respectively. Finally, the pellets were dissolved in 100 µL DNase-free water and the DNA concentration was measured by spectrophotometry with a NanoDrop 2000C (Thermo Scientific, Waltham, MA, USA). The DNA concentration was calculated as ng per mg dry weight of the leaflets.