Biology Reference
In-Depth Information
to a small aliquot of these
competent cells
(competent for transformation) and
the incubation on ice is continued for another 30 minutes. A heat shock is then
administered by putting the cells into 42°C for 2 minutes. The cells are then trans-
ferred to nutrient broth and incubated for 30-60 minutes to allow the plasmid to
express its phenotypic properties (plasmids often carry antibiotic resistance genes
as selectable markers). The cells then are plated onto agar plates containing a
selective medium. Only those bacteria that have taken up the plasmid with the
selectable marker should survive and reproduce on the selective medium.
How transformation occurs is not entirely understood. Various agents affect
the bacterial cell wall and, in the case of CaCl
2
, also may be responsible for bind-
ing DNA (the plasmid) to the cell wall. The actual uptake of DNA is stimulated
by the brief heat shock. Large DNA molecules are taken up less efficiently than
smaller DNA molecules, and the efficiency of transformation varies with the
strain of
E. coli
used. Efficiency is typically expressed as the number of transfor-
mant cells per microgram of plasmid DNA. Various protocols produce efficiencies
of 10
7
or 10
8
transformants/
μ
g of plasmid DNA.
Electroporation
also can be used to insert DNA into bacterial cells.
Electroporation involves disrupting the cell membrane briefly with an electric
current so that DNA can be incorporated. Commercial units and protocols can be
purchased for electroporation of
E. coli
.
5.11 Purifying Plasmid DNA from
E. coli
Removing the plasmids from
E. coli
is necessary if experiments are to be con-
ducted on the now-cloned DNA. The trick is to lyse (break open) the
E. coli
cells
just sufficiently so the plasmids can escape without too much contamination by
the bacterial chromosome. If the bacterial cell is lysed gently, most of the bacte-
rial chromosomal material released will be of higher molecular weight than the
plasmids and can be removed, along with the cell debris, by complexing with
detergents and high-speed centrifugation. The plasmid DNA is left in the clear
liquid remaining, and it can be extracted by one of two traditional methods.
In the first method, cesium chloride centrifugation with
ethidium bromide
(EtBr) yields bands in the centrifuge tube that contain chromosomal and plasmid
DNA at different levels due to the different densities of linear and supercoiled
DNA in the presence of EtBr.
Ethidium bromide stains DNA by intercalating between the double-stranded
DNA base pairs and in so doing causes the DNA to unwind (
Figure 5.4
).
A plasmid DNA molecule that has not been nicked is a circular double-stranded
