2.3 | DNA extraction and quantification
Bacterial cells were sedimented from 1 mL cultures by centrifugation.
The pellet was resuspended in buffer containing 0.2 M NaCl, 0.1 M
ethylenediaminetetraacetic acid (EDTA) and 15 mg/mL lysozyme, and then
incubated at 37 °C for 60 min. SST solution (20 % sarkosyl, 0.2 M NaCl,
0.5 M Tris-HCl pH 8.0, 1 mg/mL proteinase K and 4 mM dithiothreitol) was
then added and the samples were further incubated at 55 °C for 60 min.
Nucleic acids were extracted with an equal volume of phenol:chloroform
(1:1) solvent. The aqueous phase was recovered, and nucleic acids were
precipitated with 2 volumes of cold absolute ethanol in the presence of
0.3 M sodium acetate, washed with cold 70 % ethanol, dried and then
finally dissolved in buffer containing 5 mM Tris-HCl (pH 8.0) and 0.1 mM
EDTA. The concentration of DNA was determined using the
Qubit® dsDNA BR Assay Kit (Invitrogen). The integrity
of the isolated DNA was assessed by agarose gel electrophoresis.