Quantification of Extracellular DNA in Biofilms

Extracellular DNA was extracted as previously described by Kaplan et al. with modification [25 (link)]. Biofilms were grown in triplicate in TSB, TSB_glu, TSB_NaCl and TSB_CTX in 24-well polystyrene microtiter plates, in a total volume of 1 mL per well. After 24 h of growth, the liquid was carefully removed, the plates were washed once with 1XPBS (Sigma-Aldrich, St. Louis, MO, USA) and 50 μL of Tris-EDTA buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.4) (Sigma-Aldrich, St. Louis, MO, USA) was added to each well. The biofilm-forming cells were scraped off the bottom surface of the wells and were transferred to 1.5 mL microcentrifuge tubes. The tubes were centrifuged at 13,000 rpm for 25 s and 8 μL of each of the supernatants were resolved on 0.8% agarose gels. Densitometric analyses of eDNA were carried out using Image Lab software version 6.0.1(Bio-Rad Laboratories, Hercules, CA, USA).