Aim: Preparation of competent cells (DH5α Strain)

PRINCIPLE

• Competent cells are ready to use bacterial cells that possess more easily altered cell walls by which  foreign
DNA can be passed through easily.

•DNA is a highly hydrophilic molecule, normally it cannot pass through the cell membrane of bacteria so
bacteria must be made competent to take up the DNA.

•Achieved by making small holes in bacterial cells by suspending them in a solution containing a high
concentration of calcium.
 Aim: Preparation of competent cells (DH5α Strain)

Method

•Prepare a small, overnight culture of the bacteria in LB broth. Grow at 37°C without
shaking.

•About 2 h before you are ready to begin the main procedure, use 1.0 mL of the overnight
culture to inoculate 100 mL of fresh LB broth.

•This culture is grown with rapid shaking at 37°C until it reaches roughly 5 x 107 cells/ml.
Thus corresponds to an OD650 0.6-0.8, but you should calibrate this for each of your own
strains
•Take a 5 mL aliquot of each transformation reaction and transfer to sterile plastic centrifuge tubes.
Cool on ice for 10 min.

•Pellet the cells by spinning for 5 min at 5000g. It is necessary for the centrifugation to be performed
at 4°C.

•Pour off the supernatant and resuspend cells in 25 mL of cold 0.1M CaCl2. Leave on ice for at least
20 min.

•Centrifuge as in Step 3. You should observe a more diffuse pellet than previously. This is an
indication of competent cells.

•Resuspend the cells in 0.2 mL of cold 0.1M CaCl2.

•Transfer the suspensions to sterile, thin-walled glass bottles or tubes. The use of glass makes the
subsequent heat shocks more effective.
https://www.google.com/search?
q=preparation+of+competent+cells+using+calcium+chlori
de&oq=preparation+of+competent+cells&aqs=chrome.2.6
9i57j0l7.16895j1j4&sourceid=chrome&ie=UTF-
8#kpvalbx=_AsVwX-KjEoTXz7sPrIS4sAY38
 Aim: Transformation of prokaryotic model system (DH5α/BL21DE3) with cloning
/expression vector by heat shock treatment

Principle:

•Extra-chromosomal DNA will be forced to enter the cell by incubating the competent cells and the DNA together on
ice followed by a brief heat shock that causes the bacteria to take up the DNA.

•A naked DNA molecule is bound to the lipopolysaccharide (LPS) receptor molecules on the competent cell surface.

•The divalent cations generate coordination complexes with the negatively charged DNA molecules and LPS. DNA,
being a larger molecule, cannot itself cross the cell membrane to enter into the cytosol.

•The heat shock step strongly depolarizes the cell membrane of CaCl2- treated cells. Thus, the decrease in
membrane potential lowers the negativity of the cell’s inside potential which ultimately allows the movement of
negatively charged DNA into the cell’s interior.

•The subsequent cold shock again raises the membrane potential to its original value.
 Materials and Method:

For Heat-Shock Transformation

1. Thaw 50–100 µl of competent cells carefully on ice.

2. Add 1 µl of plasmid solution (concentration 1 µg/µl) to 100 µl of competent cells.
3. Incubate the cells on ice for 30 min.
4. Quickly transfer the tubes to a water bath previously set at 42 °C. Incubate for 1 min,
and then quickly transfer to ice.
5. Add 1 ml LB broth medium to the tube.
6. Incubate at 37°C for 30 min to 1 h.
7. Streak out 50–500 µl of culture onto plates containing suitable antibiotic markers.
Observation:

Successful transformation in E.coliDH5α/BL21DE3 results in appearance of bacterial colonies on

LA-Antibiotic+ plates owing to the presence of plasmid carrying antibiotic resistance marker in the
host cell.

Precautions:
•CaCl2 is a hazardous material for skin, eyes and the respiratory system and may cause burns.
Hence, use gloves while using the same.

S. Das, H. R. Dash, Microbial Biotechnology- A laboratory Manual for Bacterial Systems,

DOI 10.1007/978-81-322-2095-4_2, © Springer India 2015
https://www.youtube.com/watch?v=4CwYA4chkrU