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Preparation of the periplasmic protein fraction of E. coli by cold osmotic shock

Reagents

  • Sucrose buffer: 50 mM Tris pH 7.4, 1 mM EDTA, 20% sucrose w/v
  • 5 mM MgCl2

Method

  1. 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.
  2. Resuspend the cells in 250mL ice-cold sucrose buffer. Incubate for 10 min on ice with stirring or shaking.
  3. Centrifuge as in step 1. Remove the supernatant.
  4. Resuspend the pellet in 100mL ice-cold 5 mM MgCl2. Shake or stir for 10 min in an ice bath.
  5. 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.