Extraction of neonatal stool DNA
DNA was extracted from thawed stool samples using the QIAamp Fast DNA
Stool Mini Kit (Qiagen, Hilden, Germany). For all extractions 290-310 mg
of stool was transferred into Lysing Matrix E 2mL tubes (MP Biomedicals,
Illkirch-Graffenstaden, France). Tubes without stool were used as
negative controls for each batch of samples. Samples underwent 4 cycles
of bead beating for 30 seconds using the FastPrep-24 5G Instrument (MP
Biomedicals). The suspensions were heated to 95 degrees for 5 minutes
and then centrifuged (2 minutes, 12000 rpm). The remaining extraction
steps were performed according to the manufacturer’s instructions. DNA
was eluted using 100 μL of elution buffer and samples were centrifuged
for 2 minutes to elute the DNA. DNA yield was assessed using the Qubit
dsDNA HS Assay kit with a Qubit 2.0 fluorometer (Invitrogen). Extracted
DNA was stored at -80 degrees until amplified.