Fecal samples were collected from live mice, snap-frozen and stored at
−80°C. DNA was isolated by incubating fecal material at
65°C for 30 min in Lysing Matrix E tubes (MP Biomedicals) containing 200
mM NaCl, 100 mM Tris, 20 mM EDTA (pH 8.0), SDS and proteinase K (Qiagen).
Phenol:Choloroform:Isoamyl alcohol (Invitrogen) was added, and the samples were
homogenized at 4°C for 3 min using a bead beater homogenizer. The
samples were centrifuged at 8000 rpms for 3 min at 4°C, and the
supernatant was incubated with Phenol:Chloroform (Invitrogen) for 10 min at room
temperature. The samples were centrifuged at 13,000 rpms for 5 min at
4°C, and the aqueous phase was incubated with isopropanol and 3M sodium
acetate, pH 5.2, at −20°C for 15 hrs to precipitate DNA. The
precipitated DNA was collected by centrifugation at 13000 rpm at 4°C for
20 min, washed twice with 100% cold ethanol and resuspended in TE
buffer. The DNA was further purified using a DNeasy Blood and Tissue Kit
(Qiagen) according to the manufactures protocol.
−80°C. DNA was isolated by incubating fecal material at
65°C for 30 min in Lysing Matrix E tubes (MP Biomedicals) containing 200
mM NaCl, 100 mM Tris, 20 mM EDTA (pH 8.0), SDS and proteinase K (Qiagen).
Phenol:Choloroform:Isoamyl alcohol (Invitrogen) was added, and the samples were
homogenized at 4°C for 3 min using a bead beater homogenizer. The
samples were centrifuged at 8000 rpms for 3 min at 4°C, and the
supernatant was incubated with Phenol:Chloroform (Invitrogen) for 10 min at room
temperature. The samples were centrifuged at 13,000 rpms for 5 min at
4°C, and the aqueous phase was incubated with isopropanol and 3M sodium
acetate, pH 5.2, at −20°C for 15 hrs to precipitate DNA. The
precipitated DNA was collected by centrifugation at 13000 rpm at 4°C for
20 min, washed twice with 100% cold ethanol and resuspended in TE
buffer. The DNA was further purified using a DNeasy Blood and Tissue Kit
(Qiagen) according to the manufactures protocol.