Preparation of the periplasmic protein fraction of E. coli by cold osmotic shock
- Sucrose buffer: 50 mM Tris pH 7.4, 1 mM EDTA, 20% sucrose w/v
- 5 mM MgCl2
- Centrifuge 1L of an E. coli cell suspension for 10 min at 4C in a Sorvall GS3 rotor at 8000g to collect the cells. Discard the supernatant.
- Resuspend the cells in 250mL ice-cold sucrose buffer. Incubate for 10 min on ice with stirring or shaking.
- Centrifuge as in step 1. Remove the supernatant.
- Resuspend the pellet in 100mL ice-cold 5 mM MgCl2. Shake or stir for 10 min in an ice bath.
- Centrifuge for 10 min at 4C in a Sorvall GS3 rotor at 8000g. Save the supernatant, which is the cold osmotic shock fluid. If the supernatant is turbid, re-centrifuge and filter through a 0.2um filter.
From Grisshammer and Nagai, DNA Cloning 1.