Gene Cloning and DNA Analysis: An Introduction, Sixth Edition ...

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Bacterial culture
Figure 3.3
Harvesting bacteria by centrifugation.
Centrifuge rotor
Spin at 8000 rpm
for 10 minutes
(a) Cell lysis (b) Centrifugation to remove cell debris
Disrupt cell wall
Figure 3.4
Disrupt cell
membrane
Cell extract
Part I The Basic Principles of Gene Cloning and DNA Analysis
Cell extract
Centrifuge
Preparation of a cell extract. (a) Cell lysis. (b) Centrifugation of the cell extract to remove insoluble debris.
Pellet of bacteria
1000 ml culture at maximum cell density can then be resuspended into a volume of
10 ml or less.
3.1.2 Preparation of a cell extract
The bacterial cell is enclosed in a cytoplasmic membrane and surrounded by a rigid cell
wall. With some species, including E. coli, the cell wall may itself be enveloped by a
second, outer membrane. All of these barriers have to be disrupted to release the cell
components.
Techniques for breaking open bacterial cells can be divided into physical methods,
in which the cells are disrupted by mechanical forces, and chemical methods, where cell
lysis is brought about by exposure to chemical agents that affect the integrity of the cell
barriers. Chemical methods are most commonly used with bacterial cells when the object
is DNA preparation.
Chemical lysis generally involves one agent attacking the cell wall and another
disrupting the cell membrane (Figure 3.4a). The chemicals that are used depend on the
species of bacterium involved, but with E. coli and related organisms, weakening of
the cell wall is usually brought about by lysozyme, ethylenediamine tetraacetate (EDTA),
or a combination of both. Lysozyme is an enzyme that is present in egg white and in
secretions such as tears and saliva, and which digests the polymeric compounds that
give the cell wall its rigidity. EDTA removes magnesium ions that are essential for preserving
the overall structure of the cell envelope, and also inhibits cellular enzymes that
could degrade DNA. Under some conditions, weakening the cell wall with lysozyme or
DNA, RNA,
protein
Cell debris