Ex. No.

Genetic Transformation of Bacteria

Date:

Aim:
To transform E. coli DH 5 α competent cells using pBR322 / pARC and selection of transformed
cells.

Principle:
Bacterial transformation is the process by which bacterial cells take up naked DNA molecules. But
transfer of DNA into the most widely used bacterium, E.coli is a critical and rate limiting step in molecular
cloning techniques.

Here the bacterial cells are treated with calcium chloride and taken for heat shock that helps the
DNA to enter the cell. The bacterial cell membrane is permeable to chloride ions, but is non-permeable to
calcium ions. As the chloride ions enter the cell, water molecules accompany the charged particle. This
influx of water causes the cells to swell and is necessary for the uptake of DNA. Chilling cells in the
presence of divalent cations such as Ca2+ (in CaCl2) prepares the cell walls to become permeable to
plasmid DNA. Cells are incubated on ice with the DNA and then briefly heat shocked (42oC for 30-120
seconds), which causes the DNA to enter the cell. After this certain bacterial genes get activated this
stimulates the replication as soon as favorable conditions are provided. At this stage about 1 to 2ml of LB is
added and this mixture was incubated for 37o C for 30 minutes which is an optimum temperature for
enzymes to work.

An excellent preparation of competent cells will give ~108 colonies per μg of plasmid. A poor
preparation will be about 104/μg or less. Good non-commercial preparations should give 105 to 106
transformants per microgram of plasmid. This method has very high efficiency particularly for circular
plasmid DNAs.

Materials:
 E. coli DH 5 α competent cells, pBR322 and pARC
Ampicillin stock solution (50 mg / mL), LB broth

Method:
1. About 100 µL of competent cells were taken in a microcentrifuge tube
2. About 1 µL of plasmid DNA (~50 ng)was added
3. The mixture was incubated in ice for 30 min.
4. The transformation mix was subjected to heat shock at 42°C for 45 sec and incubated immediately
on ice for 1 min.
5. About 1 mL of LB broth with 50 ug/ mL of ampicillin and tetracycline was added and incubated at
37°C for 45 min in a shaker incubator.
6. About 100 µL of the putative transformed culture was plated on LB agar plates containing
ampicillin and tetracycline by spreading with L rod and incubated at 37°C overnight.
7. Controls: untransformed competent cells, transformed competent cells in antibiotic minus media.
8. The colonies were observed and the transformation efficiency calculated based on the formula
9. Transformation efficiency = x X 1000 / 5, where x is the number of colonies observed on plating the
given quantity of DNA (if the amount of DNA transformed is 50 ng, the amount of DNA plates is 5
ng since 100 µL of the 1 mL is plated).

Results and Discussion: