Genomic DNA extraction from blood using a classical phenol-chloroform method
A small subset of blood samples was randomly selected, and genomic DNA was extracted using a classical phenol-chloroform extraction, a rapid and inexpensive liquid-liquid extraction technique (Chacon-Cortes & Griffiths, 2014). Briefly, 200 μl of blood was lysed with 0.15% saponin at 4°C for 10 minutes and centrifuged at 2,800 g for 10 minutes. The pellet was washed with PBS, resuspended in 700 μl of Lysis buffer (10 mM Tris pH 8, 1 mM EDTA pH 8, 0.4 M NaCl and 1% SDS) and incubated at 37°C for 1 hour. A minimum of two phenol-chloroform extractions were performed by adding 700 μl of phenol-chloroform-isoamyl alcohol mixture (cat#: 77617, Sigma-Aldrich, USA) until the aqueous phase was clear. The DNA present in the aqueous phase was ethanol-precipitated and centrifuged at 9,000 g for 3 minutes at 4°C. The supernatant was discarded, and the pellet washed with 70% ethanol. The DNA pellet was resuspended in TE buffer pH 8 (1 M Tris pH 8 and 0.5 M EDTA pH 8). The concentration and purity of the extracted genomic DNA samples were measured with an IMPLEN Nanophotometer (IMPLEN, Germany).