Drummond:Periplasmic Prep: Difference between revisions
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== Preparation of the periplasmic protein fraction of <i>E. coli</i> by cold osmotic shock == | == Preparation of the periplasmic protein fraction of <i>E. coli</i> by cold osmotic shock == | ||
=== Reagents === | === Reagents === | ||
* 1 | * Sucrose buffer: 50 mM Tris pH 7.4, 1 mM EDTA, 20% sucrose w/v | ||
* 5 mM MgCl<sub>2</sub> | |||
=== Method === | === Method === | ||
# Centrifuge 1L of an <i>E. coli</i> 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 MgCl<sub>2</sub>. 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 [http://tinyurl.com/clpsox Grisshammer and Nagai, <b>DNA Cloning 1</b>]. | |||
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Latest revision as of 16:37, 27 March 2009
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
- 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.