Rnase

Homogenates from brains, hearts, livers, spleens and kidneys were used to isolate total DNA. RNase (final concentration 5 µg/µl; EURx, Poland) was added to 300 µl of the lysate and incubated at 37°C for 30 minutes. Next, thermal inactivation of RNase was performed for 10 minutes at 65°C. To samples obtained in this way, 400 µl of Tissue Cell Lysis Solution (Lucigen, USA) and 5 µl of Proteinase K (concentration 25 mg/ml; EURx, Poland) were added, and then incubated for 30 minutes at 65°C. Samples were cooled in ice for 5 minutes, then 300 µl MPC Protein Precipitation Reagent (Lucigen, USA) was added and centrifuged (8,000 x g, 10 minutes, 4°C). Five hundred µl of isopropanol (POCH, Poland) were added to the supernatant and incubated at -20°C for 24 hours. Then, the samples were centrifuged (9,600 x g, 20 minutes, 4°C), the supernatant was removed, and 700 µl of 70% ethanol (POCH, Poland) were added to the resulting colorless pellet. The samples were centrifuged (9,600 x g, 40 minutes, 4°C), the supernatant was removed, and 500 µl of 70% ethanol were added to the white pellet. The supernatant was removed, and the pellet was dried for 20 minutes under vacuum at 30°C. The pellet was suspended in 30 µl of nuclease free water (Roth, Germany) and incubated for 15 minutes at 37°C to dissolve. The obtained samples were stored at -20°C.

Yeast and yeast-like strains were cultivated in the flasks with medium containing 20.0 g/L glucose, 10.0 g/L yeast extract and 20.0 g/L peptone at 20 °C and 150 rpm. Biomass was centrifuged (5000× g, 10 min, 4 °C) and frozen at −80 °C. Next, 100 g of frozen biomass was ground in the mortar with the liquid nitrogen to a uniform powder and transferred to a 15 mL Falcon tube. One milliliter of the extraction buffer (200 mM Tris-HCl, pH 7.5; 25 mM EDTA, pH 8.0; 0.5% SDS; 250 mM NaCl) and 2 mL of the phenol:chloroform:isoamyl alcohol (25:24:1, v:v:v) mixture was added; the tube was gently shaken for 10 min at the room temperature and centrifuged at 11,000 rpm for 30 min. Water phase was collected, supplemented with 5 µL 10 mg/mL RNAse (EURx, Gdansk, Poland), incubated for 1 h at 37 °C, and extracted (equal-volume) with phenol:chloroform:isoamyl alcohol (25:24:1, v:v:v) with vigorous shaking. Water phase was collected after centrifugation (13,000 rpm, 15 min, 4 °C). DNA was precipitated by addition of 0.1 volume of 3 M sodium acetate pH 5.2 and 0.6 volume of ice-cold isopropanol. The sample was incubated over night at 20 °C and centrifuged (15,000 rpm, 15 min, 4 °C). DNA precipitate was washed twice with 70% ethanol (150 µL), centrifuged and dried for 2–3 min. DNA was dissolved in 60 µL of sterile RNAse-free distilled water.