Alkaline phosphatase buffer

Genomic DNA was extracted from the embryonic tissues of the broilers using a classical phenol/chloroform/isoamyl alcohol [25:24:1(v/v/v)] protocol. Residual RNA was treated with RNAses A and T₁ (Thermo Fisher Scientific, USA) at final concentrations of 80 μg/mL and 1500 U/mL, respectively, at 37°C for 1 h. The DNA was dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and stored at -80°C until analysis.
An improved procedure was established and performed for DNA hydrolysis [17 (link)–19 (link)]. Briefly, a 150-μL enzymolysis system was prepared as follows: approximately 20 μg of tissue DNA, one-tenth volume of 0.1 M ammonium acetate (pH 7.5), 10 μL of DNAse 1 (Thermo Fisher Scientific, USA), 15 μL of 10× DNAse 1 buffer (MgCl₂), 10 μL of FastAP Thermosensitive Alkaline Phosphatase (Thermo Fisher Scientific, USA), 15 μL of 10× Alkaline Phosphatase Buffer, 5 μL of Exonuclease 1 (Thermo Fisher Scientific, USA) and 15 μL of 10× Exonuclease 1 Buffer were mixed, and double-distilled water was added to 150 μL. The mixture was then incubated at 37°C for 4–5 h. The results of agarose gel electrophoresis showed that the genomic DNA was digested to deoxynucleosides (S1 Fig). The product was filtered through a 0.22-μm nylon membrane filter (Alltech, Deerfield, IL) and stored at -20°C until analysis.