Bio-Rad Ligation and Transformation Module - Manual (Page 27)

of the transformation (either the heat shock or the electrical pulse) and
begin to express the genes on the plasmid (such as an antibiotic resistance
gene), although this step may be omitted. The cells are plated on a selective
medium for growth, usually agar plates containing nutrient medium and the
antibiotic for which resistance is carried by the plasmid. For example, if the
plasmid contains the
amp r gene, providing resistance to ampicillin, the
agar plates should also contain ampicillin. This means that only bacteria
that have been successfully transformed and now carry the plasmid will be
able to survive and divide on the ampicillin-containing plates. The plasmid
will replicate in the bacterial cells (using the host cell’s replication machinery)
and, as the bacteria divide, the plasmids will be passed on to their offspring.
The plasmid that is used in this laboratory activity, pJet1.2 blunted vector,
contains the
ampr gene. IPTG is added to the selective medium to artificially
increase expression of the ampr gene, which is under the control of the lac
operon (normally regulated by lactose); this approach increases transformation
efficiency with this plasmid.
Even though the efficiency of bacterial transformation can be optimized by
using competent cells and by determining the best experimental conditions
for transformation and selection, transformation is still an inefficient
process, with only a small percentage of available DNA being taken into a
small percentage of the competent bacteria. After the bacteria are plated
on the selective medium, the antibiotic will prevent the untransformed cells
from growing. Transformed cells, however, will grow and divide, each
forming a colony on the plate that is the product of a single transformation
event. In other words, all the cells in each colony are clones, hence the
origin of the term cloning.
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