Faecal DNA Isolation and Purification

Faecal DNA was isolated and purified according to the literature, with minor modifications.⁴⁹ The bacterial pellet was suspended and incubated with 15 mg/ml lysozyme (Sigma-Aldrich Co., LCC) at 37°C for 1 h in TE10. Purified achromopeptidase (Wako Pure Chemical Industries, Ltd) was added at a final concentration of 2000 units/ml and then incubated at 37°C for 30 min. The suspension was treated with 1% (wt/vol) sodium dodecyl sulphate and 1 mg/ml proteinase K (Merck Japan) and incubated at 55°C for 1 h. The lysate was treated with phenol/chloroform/isoamyl alcohol (Life Technologies Japan, Ltd). DNA was precipitated by adding ethanol and pelleted by centrifugation at 3,300 g at 4°C for 15 min. The DNA pellet was rinsed with 75% ethanol, dried, and dissolved in 10 mM Tris-HCl/1 mM EDTA (TE). DNA samples were purified by treating with 1 mg/ml RNase A (Wako Pure Chemical Industries, Ltd) at 37°C for 30 min and precipitated by adding equal volumes of 20% polyethylene glycol solution (PEG6000-2.5M NaCl). DNA was pelleted by centrifugation at 8,060 g at 4°C, rinsed with 75% ethanol, and dissolved in TE.