Bacterial DNA Isolation from Milk

Milk samples (5–10 mL) were thawed and centrifuged at 4000 × g for 20 min to separate fat and cells from whey. Thereafter, total DNA was isolated from the pellets by using the MasterPure Complete DNA and RNA Purification Kit (Epicenter) according to the manufacturer's instructions with some modifications (Simón-Soro et al., 2015 (link)). Two hundred and fifty microliters of saline solution and 250 μl of lysis buffer were added to the pellets, together with Pathogen Lysis Tubes (QIAGEN) glass beads. Both chemical and physical cells disruption was performed after mixing vigorously the samples in a TissueLyser II (QIAGEN) during 5 min at 30 Hz, incubating in dry ice 3 and 5 min at 65°C in a thermoblock, repeating the process 2 times. Fifty microliters of lysozyme (20 mg/ml) and 5 μl of lysostaphin (20 μg/ ml) were added to the tubes, and the samples were incubated for 1 h at 37°C. Two microliters of proteinase K were added and samples were incubated for 15 m at 65°C. The reaction was ended putting tubes on ice, and proteins were precipitated using 350 μl of the protein precipitation agent, discarding the pellets. DNA was precipitated using isopropanol, washed with 70% Ethanol and resuspended with 30 μl TE buffer. The total DNA isolated was quantified with a NanoDrop ND-1000 (ThermoScientific) spectrophotometer.