Minilysate

(AMS procedure)

  1. Grow cells in 3 ml culture (~ 12 hrs).
  2. Transfer to Eppendorf tube.
  3. Spin 2 min
  4. Remove supernatant.
  5. Resusupend pellet of cells in 400 ml STET.
  6. Add 40 ul lysozyme sol.
  7. Vortex
  8. Incubate on ice 15 min.
  9. Incubate in 95o C to 100o C water for 90 sec.
  10. Centrifuge 30 min in cold room.
  11. Fish out pellet.
  12. Add 440 ul isopropanol.
  13. Mix and let stand ~ 5 min. at room temp.
  14. Spin 15 min.
  15. Wash 2X with 70% EtOH.
  16. Dry and resuspend in 50 ul TE.


STET:

8% Sucrose

100mM EDTA

5% Triton X-100

50 mM EDTA

50 mM Tris HCl (pH 8.0)

Lysozyme:

10 mg/ml in water.

TE:

10 mM Tris HCl (pH 7.0)

1 mM EDTA