Qiafilter midi kit

Plasmid DNA for Southern blotting was extracted from exponential BHI cultures (optical density at 600 nm [OD₆₀₀] ≈1.0) using an alkaline precipitation protocol as previously described (7 (link)). Total and plasmid-enriched DNA for sequencing was obtained using the GenElute bacterial genomic DNA kit (Sigma-Aldrich) and QIAfilter midikit (Qiagen), respectively. Total DNA for routine PCR tests was prepared by heating single bacterial colonies at 100°C in 100 μl of ultrapure water and centrifugation for 90 s at 16,000 × g. PCR was performed as previously described (13 (link)). Oligonucleotide primers used are shown in Table S4. For Southern blotting, plasmid preparations were electrophoresed in 0.5% agarose and transferred to positively charged nylon membranes (Roche) after treatment of the gels with denaturing solution (1.5 M NaCl 0.5 N NaOH) for 30 min and neutralizing solution (1.5 M NaCl, 0.5 M Tris-HCl pH 7.5) twice for 20 min. Probes consisted of digoxigenin-labeled internal fragments of the genes of interest [vapA, erm(46), and IS6100 transposase] generated with the PCR DIG probe synthesis kit (Roche) using suitable oligonucleotide primers (Table S4). Membranes were hybridized at high stringency and developed according to the manufacturer’s instructions. Hybridized membranes were stripped with 0.2 N NaOH–0.1% SDS at 45°C for 30 min before reprobing.

Beginning at day 21 of life, faecal pellets were collected daily for the first weeks of life, and weekly in mid- and late-life, then stored at −20 °C until processing. DNA was extracted from pellets using the PowerSoil DNA isolation kit (Mo Bio Laboratories Inc., Carlsbad, CA) and stored at −20 °C. qPCR was performed in duplicate, using the SYBR Green 1 Master Kit on a LightCycler 480 (Roche Applied Science, Indianapolis, IN). Bacterial standards were cloned into pGEM-T Easy vector following standard procedures and purified using plasmid purification QIAfilter Midi Kit (QIAGEN Inc., Valencia, CA), using described protocols and primers for total bacteria, Bacteroidetes and Firmicutes47 (link). For fixed time point statistical comparisons, a Shapiro–Wilk normality test was first performed on each qPCR variable for each group. Two-sided t-tests and Wilcoxon Rank-Sum tests were used for group comparisons on the normal and non-normal data, respectively.