Boiling?lysis?mini-prep

1、Grow an overnight culture from a single colony in 2 mls LB + antibiotic.

2、Transfer 1.5 mls of culture to eppendorf tube.Spin for 1 minute on high and remove supernatant.

3、Add 700 µl STET. Add 25 µl lysozyme stock solution.Vortex to resuspend pellet.Place tubes on ice for 5-10 minutes.

4、Boil tubes for 1 minute.

5、Spin in microfuge for 10 minutes.

6、Pull out snot pellet with a toothpick.

7、Add 700 µl of room temperature isopropanol.Mix tubes by inversion.

8、Spin in a microfuge for 10 minutes.A small white/clear pellet will form.

9、Remove supernatant.Wash with 70% ethanol.

10、Dry pellet and resuspend in 50µl water containing 20 µg/ml RNase A.

STET
      8% sucrose
      5% Triton X-100
      50 mM Tris-HCl,pH 8.0
      50 mM EDTA,pH 8.0
      store at 4°C

Lysozyme Stock Solution (10 mg/ml)
      150 mg lysozyme
      3.75 mls 1M Tris-HCl,pH 8.0
      11.25 mls H2O
      store at -20°C