Histiocyte lysis buffer

For all samples, genomic DNA was extracted from 100 mg powder using the phenol/chloroform method [19 ]. Briefly speaking, 800 μL histiocyte lysis buffer (Tiangen, China) with 100 μg proteinase K (Tiangen, China) was added to each sample, vortexed, and incubated at 65°C for 60 min with occasional vigorous shaking; an equal volume of phenol/chloroform was added, mixed, and centrifuged at 12000 rpm for 10 min. The aqueous (upper) layer was transferred to a clean tube; an equal volume of chloroform was added, mixed, and then centrifuged for 5 min at 12000 rpm. The aqueous (upper) layer was transferred to a clean tube; a one-tenth volume of 3 M Na acetate (pH 5.2) and two volumes of ice-cold EtOH (100%) were added, mixed, and incubated at −20°C for 30 min and then centrifuged at 12000 rpm for 30 min at 4°C. The supernatant was removed and the DNA pellet was washed twice with 75% EtOH and centrifuged at 12000 rpm for 2 min at 4°C; the supernatant was removed and the pellet was air-dried for 30 min at room temperature, resuspended in 100 μL ddH₂O, and stored at −20°C.

DNA was extracted from meat samples by phenol/chloroform extraction [16 (link)]. All samples (100 mg) were mixed with 800 μl histiocyte lysis buffer (TIANGEN Biotech, Beijing, China) and 100 μg proteinase K (TIANGEN Biotech, Beijing, China). Following 60 min incubation at 65°C with occasional vigorous shaking, an equal volume of phenol/chloroform was added; samples were mixed and centrifuged at 12,000 rpm for 10 min. The resulting supernatant was collected, and an equal volume of chloroform was added; samples were mixed and centrifuged at 12,000 rpm for 5 min. The aqueous layer was transferred to a clean tube. An equal volume of ice-cold 100% EtOH and a one-tenth volume of 3M sodium acetate (pH 5.2) were added; samples were mixed incubated at -20°C for 30 min, and centrifuged at 12,000 rpm for 30 min. The supernatant was discarded and the pellet was washed twice with 800 μl 75% EtOH. Following centrifugation at 12,000 rpm for 5 min, the pellet was air-dried and resuspended in 100 μl DNAse-free and RNAse-free water (Invitrogen, Carlsbad, CA, USA). The DNA concentration of each sample was measured in a NanoVue spectrophotometer (GE Healthcare China, Beijing, China).