Pellet 1.5 ml of an overnight culture at 12,000 rpm in Eppendorf centrifuge at RT for 3 min.
Resuspend bacterial pellet in 350 µl of STET buffer.
Add 25 µl of freshly prepared solution of lysozyme (10 mg/ml in 10 mM Tris-Cl, pH 8).
Mix by vortexing for 3 sec.
Place the tube in a boiling water bath for exactly 40 sec.
Centrifuge the lysate at 12,000 g for 10 min at room temp.
Remove the glob of bacterial debris with a sterile toothpick.
Add to the supernatant 40 µl of 2.5 M NaOAc (pH 5.2) and 420 µl of isopropanol.
Mix by vortexing and store the tube for 5 min at room temp.
Centrifuge at 12,000 g for 5 min at 4℃.
Remove the supernatant and wash with 1 ml 70% ethanol.
Dry the pellet and resuspend in TE (pH 8) with RNase A (20 µg/ml).
STET buffer:
0.1 M NaCl
10 mM Tris-Cl, pH 8.0
1 mM EDTA, pH 8.0
5% Triton X-100
