MINI?PREP?BY?BOILING

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