Rnase free molecular biology grade water

Biopsy specimens were thawed on ice before being cut into approximately 30-mg sections. One section was added to a MACs M tube (Miltenyi Biotech, UK) containing 1 ml Qiazol (Qiagen, UK) and dissociated on a GentleMACs instrument (Miltenyi Biotech, UK) using the manufacturer’s RNA settings. The sample was centrifuged and incubated at room temperature (RT) for 5 min before transferring the lysate to a 1.5-ml centrifuge tube. Proteinase K (20 μl) was added to the sample before being incubated at 56 °C for an hour. Chloroform (200 μl) was added, and the mixture was shaken vigorously for 15 s. The sample was then incubated at RT for 2 min before being centrifuged at 12,000 × g for 15 min at 4 °C. The upper aqueous phase was transferred to a fresh 1.5-ml centrifuge tube before the addition of a 1× volume of 70 % ethanol. The sample (up to 700 μl) was added to an RNeasy minispin column (Qiagen, UK) and centrifuged at RT at 8,000 × g for 30 s. Any remaining sample was also passed through the column. All remaining steps were performed according to the manufacturer’s guidelines, with elution in 30 μl of RNase-free molecular-biology-grade water (Thermo Fisher, UK).

The interdigital space swabs were placed on a MixMate instrument (Thermo Fisher, UK) for 5 min at 800 rpm to thoroughly disperse the bacteria in the Amies solution from the swab. The liquid was transferred into a low-bind 1.5-ml tube and centrifuged at 12,000 rpm for 5 min. The supernatant was removed, and the pellets were resuspended in 200 μl of RNase-free molecular-biology-grade water (Thermo Fisher, UK) (52 (link)). DNA was isolated using the Qiagen Cador pathogen minikit, according to the manufacturer’s guidelines, and eventually eluted in 60 μl of elution buffer. The DNA samples were quantified using the Qubit 3.0 system and double-stranded DNA (dsDNA) high-sensitivity dye (Qiagen).