Competence is ability of bacteria to take up foreign DNA. Transformation is alteration of genetic material resulting from uptake of exogenous genetic material from surrounding environment through cell membrane .

1. Practical#12
2-01-2019
Preparationof competent cells and transformation
Competence:
It is the ability of bacteriato take up foreign DNA from the
environment.It can be natural or induced. Competent cells
have altered Cell walls so that they can easily incorporate
foreign DNA. Cells can be made competent either by
chemical methods usinga series of coldsaltwashes orby
electrocompetentmethods wherethe cells are chilled and
washed with cold deionizedwater and 10% glycerol in the
absence of high salt envioronment.
Principle:
Bacterial cells are grown to logarithmic phase and
harvested. Cells growing exponentially can be rendered
more easily than at other stages of growth. After
harvesting the cells are treated with different methods in
order to disrupt the thereby preparing the cells to accept
plasmid DNA.
Requirements:
LB broth, culture plates, 1M cacl2.2h20(ice cold),MgCl2
Cacl2(icecold), shakingincubator, Centrifuge, Inoculation
loop,Microfuge tubes,polypropylene tubes, pipettes,tips.

2. Procedure:
1.Take an ioculation loop,sterilize it by flaming it utill
red hot and allow to cool for sometime.
2.Pick a single bacterial colony from plate and transfer
the colony into LB broth.
3.Incubate the culture at overnight at 37 in shaking
incubator.
4.Take a microlitre micropippete and insert fresh
autoclaved tip,transfer 1 mL of the overnight culture
to 100 mL LB broth and incubate for 3 hours at 37 C
in a shaking incubator.
5.Transfer the bacterial cells to ice cold sterile
polypropylene tubes store them on ice for 10mins.
6.Centrifuge the tubes at 4100 rpm for 10 mins at 4 C.
7.Decant the medium from the cells pellete and 30 mL
of ice colg cacl2-Mgcl2 solution.
8.Resuspend the pellete by gentle swirling vortexing in
30 mL of cacl2-Mgcl2.
9.Centrifuge the tubes at 4100 rpm for 10 mins at 4 C.
10. Decant themedium and add 2mL ofice cold 0.1M
cacl2 Solution and repeat resuspension step.
11. Aliquot 100 microlitres of cells into
microcentrifuge tubes placed on ice.
12. Competent cellscan be used for transformation or
stored at –70 C.

3. Transformation
Transformation is the genetic alternationof a cell resulting
from the direct uptake and incorporation of exogenous
genetic material from its surroundings through the cell
membrane. There are two primary methods for
transforming bacterial cells:
1.Heat shock : The plasmid-cell mixture is briefly heated
to 45-50 C allowing the DNA to enter the cell through
the disrupted membrane and then placed on ice.
2.Electroporation: The plasmid-cell mixture is exposed to
an electrical current , openingpores in the membrane so
that the plasmid can enter the cells.
Principle:
Plasmid DNA is mixed with chilled cells and incubate on
ice to allowtheplasmid to come into close contact with the
cells. The plasmid-cell mixture is treated either with heat
shock or electroporation method allowing the DNA to
enter the competent cells. This mixture is then placed back
on ice to retain the plasmids inside thebacteria. These cells

4. maintain integrity to keep the plasmids and when
medium is added recover and divide.
Requirements:
Shakin incubator,Stationary incubator,Water bath, Ice
bucket filled with ice,microcentrifuge,sterile spreading
device,LB agar plates with antibiotics,Competent
Cells,Insert DNA to be transformed.
Procedure:
1.Take competent cells out of –80 C and keep them on
ice.
2.Remove agar plates and incubate at 37 C before use
and then incubate at room temperature.
3.Mix the competent Cells with that of the DNA in a
microcentrifuge or falcon tube.Gentlymix it 3-4 times
and incubate it on ice.
4.Heat shock the samples by putting the cells on water
bath for 30-60 seconds after that put it again on ice.
5.Place the bacterial cell on LB broth at shaking
incubator for 45mins.
6.Transfer the cultureson agar plates and incubateat 37
C.

5. Calculationof TransformationEfficiency:
The transformation efficiency is defined as the number of
transformantsgeneratedper µg of supercoiledplasmid
DNA used in the transformation reaction.
Transformation efficiency is calculated using the formula
below:
Number of Colonies on Plate (df) / Amount
of DNA plated(ng) x 1000ng/µg

6. Applications of Transformation:
 to make multiple copies of the DNA
 in cloning procedures
 to express large amounts of proteins and enzymes
 in the generation of cDNA libraries
 in DNA linkage studies