1. Incorporation of Ethidium Bromide allows the visualisation of DNA molecules:

2. DNA molecules from 2 different species, if cleaved by the same
restriction enzyme, will join, to form a recombinant molecule.

3. Why do you need to clone genes?
In order to manipulate DNA, you need a reasonable quantity
of a specific gene (1 ug)
2) There might be just ONE gene (one molecule) coding for the
protein you are interested
3) Gene cloning allows you to produce multiple copies of any
gene - milligramme amounts of individual genes are produced.
4) This quantity of DNA is needed for DNA manipulations
restriction, ligations, fragment purification etc…

4. A fragment of DNA containing the gene to be cloned is inserted
into a circular DNA molecule called a PLASMID.
B) The plasmid acts as a VEHICLE or VECTOR to transport the gene
to be cloned into a host cell (bacterium, plant, animal)
C) Within the host cell, the vector multiplies, producing multiple copies
of itself (and of the gene it carries)
D) Division of the host cell results in copies of the recombinant
molecule passing to the progeny.
E) After a large number of divisions, a colony of identical cells is
produced, each of which contains the recombinant plasmid.
F) Plasmid can be purified from the bacterial cells.

5. Some facts about PLASMIDS….

6. 1) Circular DNA molecules found in bacterial cells
2) “Natural” molecules – replicate independently of the bacterial
chromosome.
3) Exist to allow the bacterium to survive in a changing environment
4) Usually carry genes which code for antibiotic resistance

7. Only 1% of the cells in a bacterial population might carry a plasmid
containing an antibiotic resistance gene.
If the bacterial population is exposed to the antibiotic, 99% of the
cells will be killed, but 1% will survive.
Under selective pressure these 1% will survive and divide.
Antibiotic resistance (plasmid borne) spreads rapidly through
bacterial population.

8. Plasmids, therefore, are DNA molecules which allow bacteria
To adapt to rapidly changing environments.
Plasmids can be exchanged between
bacteria by mating (conjugation)
Almost all major bacterial diseases are now
resistant because of the widespread misuse of antibiotics
COMPLETE THE ANTIBIOTIC DOSE – TAKE ALL TABLETS!

9. Naturally occurring plasmids have been extensively modified for use
as cloning vectors.
Small
Contain genes for antibiotic resistance
Have restriction enzyme sites to
allow the cloning of a wide
range of DNA fragments.
Contain an origin of replication
Carry a B- galactosidase gene (lacZ)
gene to allow the selection of
recombinants

10. Cloning a human gene into a bacterial plasmid:
Plasmid and gene to be cloned must generate compatible termini
upon cleavage by the restriction enzyme.

11. Transformation of a recombinant plasmid into competent cells will
give rise to the following types of cells:
1) Cells which have not taken up a plasmid – non-transformants
2) Cells which have taken up a non-recombinant plasmid
-transformants
3) Cells which have taken up recombinant plasmids - recombinants
4) Cells which have taken up the recombinant plasmid carrying
the gene you are after.
5) We need to be able to distinguish between these 4 possibilities

12. It is relatively easy to distinguish between these 4 possibilities:
Cells which have not taken up a plasmid – non-transformants
Will not grow on antibiotic containing plates.
Cells which have taken up a recombinant plasmid
-transformants can be identified using an enzymatic
insertional inactivation system