CTAB-Based Infant Stool DNA Extraction

Stool samples from 308 infants were extracted using a modified cetyltrimethylammonium bromide (CTAB) buffer based protocol^{13 (link)}. Briefly, 0.5 ml of modified CTAB extraction buffer were added to 25 mg of stool in a 2 ml Lysing Matrix E tube (MP Biomedicals, Santa Ana, CA) then incubated (65 °C, 15 min). Samples were bead–beaten (5.5 m s⁻¹, 30 sec) in a Fastprep–24 (MP Biomedicals, Santa Ana, CA) followed by the addition of 0.5 ml of phenol:chloroform:isoamyl alcohol (25:24:1). Following centrifugation (14,000 rpm, 5 min), the supernatant was added to a heavy phase–lock gel tube (5 Prime, Gaithersburg, MD) and chloroform (v:v) was added. Samples were centrifuged (14,000 rpm, 5 min); resulting supernatants were added to fresh tubes, followed by addition of 1 μl of linear acrylamide prior to PEG–NaCl (2v:v). Samples were incubated (21 °C, 2 h), washed with 70% EtOH and resuspended in 10 mM Tris–Cl, pH 8.5.