Bio-Rad Ligation and Transformation Module - Manual (Page 25)

• Heat shock is the most easily accomplished transformation method, as
it does not require any equipment other than a water bath. Plasmid
DNA and heat-shock competent cells in calcium chloride are mixed
together and incubated on ice for several minutes. Although the
mechanism is not fully understood, calcium chloride causes DNA to
bind to the bacterial cell wall. The cells are then subjected to a brief
heat shock resulting in the uptake of DNA into the bacteria. Traditionally
bacteria are heat shocked by incubation at 42°C for 50 sec. However in
this laboratory the bacteria are heat shocked by spreading the ice cold
bacteria directly onto warm agar plates at 37°C. Cells intended for heat
shock transformation must be in the exponential growth phase to be
highly competent
• Electroporation is also commonly used for transformation, and its
mechanism of enabling DNA uptake is somewhat better understood
than heat-shock transformation. When bacterial cells are subjected to a
brief electrical shock, small pores open in their cell wall, allowing DNA
to enter the cells. For electroporation, electrocompetent bacteria and
plasmid DNA are mixed and placed in a special type of cuvette, a square
test tube with metal electrodes on two sides (see figure on page 22).
The cuvette is placed in an instrument called an electroporator that
delivers an electrical charge of specific strength and duration to the
cells. The electricity travels through the cells between the two electrodes,
which is why electrocompetent cells must be prepared in a solution of
very low ionic strength. For electroporation to be successful, the cells
themselves must carry the current across the gap between the electrodes.
If there are many ions (like Na
+ ) in the solution, the ions will carry the
current instead of the cells, causing the cells to overheat and die
21