Nanodrop 8000 spectrophometer

A protocol for DNA extraction in barley (Brandes, in prep.) was slightly modified for DNA extraction in caraway. 192 plants per each F 1 population (n = 7) were grown in greenhouse in 96 pot plates. The youngest developing leaf per plant (BBCH = 15-18 (Hack et al. 1992 )) was sampled. Leaves were collected in racked collection microtubes (Cat. no. 19560, Qiagen) . Two 4 mm steel beads per sample were added. Samples were frozen with liquid nitrogen. Samples were ground using mixer mill MM 300 (Retsch). Samples were incubated at 65 °C for 45 min after adding 600 ll extraction buffer (1 M guanidine thiocyanate, 2 M NaCl, 30 mM NaAc) per sample. Samples were spun using Rotina 420 centrifuge (Hettich) for 30 min at 4000 rpm. 400 ll of liquid per sample were transferred to new collection microtubes and 2.5 ll RNase A (5 mg/ml) was added. Samples were incubated for 30 min at 35 °C. DNA was precipitated, adding 300 ll propan-2-ol per sample. Samples were spun using Rotina 420 centrifuge (Hettich) for 30 min at 4000 rpm. DNA pellets were washed in two steps, adding 600 ll per solution and sample (wash solution 1: 76% ethanol, 200 mM NaAc; wash solution 2: 76% ethanol, 10 mM NaAc). Dried DNA was dissolved in 600 ll TE-buffer (10 mM Tris/HCl, 1 mM EDTA) per sample. DNA was quantified using nanodrop 8000 spectrophometer (Thermo Fisher Scientific).