Genomic DNA extraction. Ten (10) mL of the bacterial culture
in Tryptic Soy Broth (TSB, Merck®, USA) was incubated at 37 C
for 24 h followed by centrifugation at 3500 g for 15 min. The
pellet was resuspended in 500 mL TE buffer (1.21 g Tris þ 0.37 g
EDTA/100 mL distilled water) and centrifuged again at 14000 g
for 1 min. The pellet was resolved in SET tampon (0.788 g Tris HClþ
1.86 g EDTA þ 20 g sacharose/100 mL distilled water) which contains
5 mg/mL lysozyme and incubated at 37 C for 45 min. TE
tampon (200 mL), 20% SDS (30 mL), and NaCl (5 M,100 mL)was added
to the cell suspension and mixed thoroughly. In order to provide an
emulsion, phenol was added to the mixture. The mixture was
centrifuged at 12000 g for 15 min, and the upper phase was
transferred into a new tube. Chloroform/isoamylalcohol was added
and centrifugation step was repeated using the same conditions.
Pure ethanol was added to the tube and centrifuged for the last
time. The pellet was air dryed and resuspended in TE tampon.