Drummond:Periplasmic Prep

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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. }}