Gene Cloning

What is Cloning
• Cloning is the process of producing
individuals with identical or virtually
identical DNA, either naturally or artificially.
In nature, many organisms produce clones
through asexual reproduction.
• Genome Cloning:
• Using nuclear DNA from one organism to
create a second organism with the same
nuclear DNA.
Cloning
Cloning
• Molecular Cloning:
• The replication of one part of DNA to
produce a population of cells with identical
DNA molecules.

• Cell Based
• PCR
What are cloning vectors?
DNA molecules that are used to "transport" cloned
sequences between biological hosts and the test tube.

1. Autonomous Replication.
2. Dominant Genetic marker for selection.
3. Unique Restriction sites.
4. Minimum amount of nonessential DNA to optimize
cloning.
Bacterial Plasmids
 Activity Plan
• 1- Gene of interest
• 2- Preparation of Competent Cells
• 3- Ligation of DNA into Vector
• 4- Transformation
• 5- Growth on Agar plates
• 6- Screening of Interested Gene
Closing of Plasmid
1- Restriction Digestion of
DNA
Restriction Sites
Restriction Digestion of
Plasmid Vector
Ligation
Transformation
• Exogenous DNA into cells (E. coli)
• Two methods of Transformation

a- Chemical method using CaCl2

B- Electroporation
Transformation: CaCl2
Transformation: Electroporation
What did the cells take up?
 Growth on Agar plates
 Blue colonies represent Ampicillin-resistant
bacteria that contain Vector and express a
functional fragmen, (beta galactosidase) from
an intact LacZ coding sequence.

betagalactosidaze+x-gal= blue coloured product

White colonies represent Ampicillin-resistant

bacteria that contain Insert and do not produce
LacZ, fragment.
• IPTG: Inducer of LacZ
• X-gal: Analogue of Lactose
Insulin Production by Cloning