Ex. No.

Preparation of competent cells of E. coli

Date:

Aim:
Preparation of competent cells of E. coli DH5α using the CaCl2 method.

Principle:
Competence is the ability of a cell to take up extracellular ("naked") DNA from its environment.
Competence is distinguished into natural competence, a genetically specified ability of bacteria that is
thought to occur under natural conditions as well as in the laboratory, and induced or artificial competence,
arising when cells in laboratory cultures are treated to make them transiently permeable to DNA.
Competent cells are those that possess more easily altered cell walls that DNA can be passed
through easily. These cells readily incorporate foreign DNA. On example of a competent cell is E. coli. A
cell that is not already competent can be made more competent through calcium chloride (a substance that
creates organic ions) and heat shock. This is especially easy with cells undergoing very rapid growth. This
characteristic has an interesting potential application. With low levels of calcium chloride and heat shock
that does not affect less-susceptible cells (in this case, those that do not have very rapid growth), it may be
possible to make rapidly-dividing cancer cells competent enough to take up DNA that will replicate with the
cell. This DNA can have a variety of different characteristics: it may be easily traceable, or it may be very
susceptible to a specific chemotherapy drug, for instance.
Since DNA is a very hydrophilic molecule, it won't normally pass through a bacterial cell's
membrane. In order to make bacteria take in the plasmid, they must first be made "competent" to take up
DNA. This is done by creating small holes in the bacterial cells by suspending them in a solution with a high
concentration of calcium. DNA can then be forced into the cells by incubating the cells and the DNA
together on ice, placing them briefly at 42° C (heat shock), and then putting them back on ice [snap
cooling]. This causes the bacteria to take in the DNA. The cells are then plated out on antibiotic containing
media.The calcium chloride ions neutralize the repulsion between the negatively charged phospholipid
heads of the cell membrane and the negatively charged phosphate groups on the DNA
The procedure to prepare competent cells can sometimes be tricky. Bacteria aren't very stable
when they have holes put in them, and they die easily. A poorly performed procedure can result in cells that
aren't very competent to take up DNA. A well- performed procedure will result in very competent cells. The
competency of a stock of competent cells is determined by calculating how many E. coli colonies are
produced per microgram of DNA added. An excellent preparation of competent cells will give ~108 colonies
per µg. A poor preparation will be about 104 / µg or less.

Materials:
Luria Bertani broth
Plasmid DNA
Calcium chloride solution (0.1 M):
Prepared by dissolving 1.47 g of calcium chloride in 100 mL of distilled water. Sterilized before use
for competent cell preparation.
Calcium chloride (0.1 M) and 20% glycerol solution:
Prepared by dissolving 1.47 g of calcium chloride in 50 mL of distilled water. About 20 mL of
glycerol was added and volume made up to 100 mL by distilled water. Sterilized before use.
Luria Bertani medium:
Tryptone – 10 g, yeast extract – 5 g, sodium chloride – 10 g were dissolved in 200 mL of water.
The pH was adjusted to 7.0 and volume made up to 1000 mL. Sterilized by autoclaving.

Method:
1. The E. coli culture was streaked on a LB plate and incubated overnight at 37°C.
2. Single, well isolated colony was selected and transferred to 5 mL of LB broth and incubated
overnight at 37°C in a shaker incubator.
3. About 1 mL of the saturated overnight culture was transferred to 100 mL LB broth and incubated at
37°C in a shaker incubated until the OD at 600 reached 0.5 (2-2.5 h). Sterile LB was used as blank
to set zero in the spectrophotometer. The OD was checked frequently to avoid overgrowth.
4. The culture was transferred to centrifuge tube and centrifuged at 4000 rpm for 5 min at 4°C.
5. The supernatant was discarded immediately and the pellet resuspended in 10 mL of ice-cold 0.1 M
CaCl2.
6. The tubes were incubated in ice for 30 min.
7. Centrifuged at 4000 rpm for 5 min at 4°C.
8. The pellet was resuspended in 1 mL of 0.1 M CaCl2 containing 20% glycerol.
9. The compentent cells were aliquoted 150 µL in 1.5 mL microcentrifuge tubes.
 (If they are not immediately used, cells are stored at 4 oC for maximum of 6 h without significant
loss of competency. The same competent cells can also be stored at -70 oC for long-term storage).

Results and Discussion: