Phenol chloroform isoamyl alcohol

E. coli strains were cultured on Tryptone Soya broth (TSB; Oxoid CM 0129) at 37 °C for 24 h. In total, 750 µL from this enrichment culture broth was transferred into Eppendorf tubes (2 mL) and centrifuged at 10,000 rpm for 5 min. Then, the bacterial pellet was resuspended in TE (10 mM Tris-HCl pH 8.0 and 1 mM EDTA) containing lysozyme (Sigma 7651) and incubated at 37 °C for 18 h. In the next stage, 250 µL of 10% SDS and 20 µL of 20 mg/mL proteinase K (Sigma P2308-100MG)/dH₂O were added into the Eppendorf tube, and the tubes were incubated at 56 °C for 2 h. Then, 750 µL of phenol/chloroform/isoamyl alcohol (25:24:1) (Amresco K169, Solon, OH, USA) was added, and the tubes were centrifuged at 14,000 rpm for 15 min. The supernatant was transferred into another Eppendorf tube. First, 150 µL of 5 M NaCl and then 700 µL of 2-propanol (Merck 1096342511, Darmstadt, Germany) were added for precipitating DNA, followed by centrifugation at 14,000 rpm for 10 min. Then, the supernatant was removed, and the pellet was washed twice with 1 mL of 80% cold ethanol (Sigma 459844) and centrifuged at 14,000 rpm for 10 min [19 (link)]. The pellet was resuspended in 75 µL of ultra-pure water. The acquired DNA was tested by BioTek Epoch2 (Agilent, Santa Clara, CA, USA) for its quality and stored at −20 °C.

DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) sections of heart and parotid gland tissues using the QIamp DNA FFPE tissue kit according to the manufacturer’s instructions. For genomic DNA isolation from fresh tissues, harderian and parotid gland tissues were minced in 1 ml of PBS, transferred to microcentrifuge tubes, and centrifuged for 5 min at 6,000 × g. The tissue pellets were incubated in 0.5 ml of lysis buffer (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 25 mM EDTA, pH 8.0, 1% SDS) with proteinase K (Promega) added to a 0.5-mg/ml final concentration overnight at 56°C with gentle shaking. This tissue solution was extracted with an equal amount of phenol chloroform-isoamyl alcohol (25:24:1; Amresco) for 10 min at room temperature and centrifuged for 5 min at 16,000 × g (Eppendorf 5424R). The aqueous layer was transferred into a new tube and mixed with 0.5 vol of 7.5 M ammonium acetate, 2 vol of 100% ethanol. After centrifugation for 5 min at 16,000 × g, the DNA pellet was rinsed with 70% ethanol, air dried, and subsequently dissolved in Tris-EDTA (TE) buffer.