Bio-Rad Ligation and Transformation Module - Manual (Page 26)

Bio-Rad electroporation cuvette.
There are ways to increase the number of competent cells in a bacterial
culture. To prepare competent cells for heat shock transformation, the
bacteria must be washed to remove the growth medium, then resuspended
in an ice cold calcium chloride solution. For electroporation, the cells must be
washed repeatedly in chilled buffer, and resuspended in a chilled sterile
solution that has very low ionic strength. In both cases, the cells must be
in solution and at a high concentration for transformation to be successful
and the cells must be kept cold at all times prior to transformation. The
cells are extremely fragile at this stage and the cold temperature keeps
them inert. If they are warmed up in transformation solution, they will start
to die. The transformation protocol also requires the cells to be cold; even
though the exact process of transformation is still not fully understood, it is
presumed that the cold hardens the cell membranes of the bacteria and
the heat shock rapidly melts or breaks the membranes, which allows the
DNA inside the cells.
Since bacteria have defense mechanisms that use restriction enzymes to
degrade foreign DNA, only mutant strains that no longer have restriction
activity can be used for transformation. Normal bacteria would degrade the
plasmid DNA as soon as it enters the cell. Mutant strains for transformation
are widely available, for example, the
E. coli HB101 K-12 strain recommended
for use in this activity.
What Happens After Transformation
After either transformation method, the cells are usually incubated in
nutrient medium for up to 1 hour to allow them to recover from the stress
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Chamber for cells
Metal electrodes