This is a cached page for the URL (http://cancer.ucsd.edu/howelllab/Mini%20Prep%20by%20Boiling.html). To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
MINI PREP BY BOILING

MINI PREP BY BOILING
  1. Pellet 1.5 ml of an overnight culture at 12,000 rpm in Eppendorf centrifuge at RT for 3 min.
  2. Resuspend bacterial pellet in 350 µl of STET buffer.
  3. 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.
  4. Place the tube in a boiling water bath for exactly 40 sec.
  5. Centrifuge the lysate at 12,000 g for 10 min at room temp.
  6. Remove the glob of bacterial debris with a sterile toothpick.
  7. 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.
  8. Centrifuge at 12,000 g for 5 min at 4°C.
  9. Remove the supernatant and wash with 1 ml 70% ethanol.
  10. 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