Plasmids are circular, self-replicating DNA molecules that are commonly used as cloning vectors. Key properties of plasmids that make them useful for cloning include their ability to replicate independently of the bacterial chromosome, carry genes for antibiotic resistance or other selectable markers, and accept foreign DNA at specific restriction sites. Common plasmids used for cloning are pBR322, which has features like a high copy number and blue-white screening, and pUC plasmids, which have even higher copy numbers. Plasmids can shuttle DNA between bacterial and yeast cells, and RNA production plasmids contain promoters to transcribe cloned DNA into RNA for applications like Northern blotting.

1. Cloning Vectors
Plasmid

2. Cloning can provide a pure sample of an individual DNA
fragment or gene ,separated from all the other genes in the
cell.
Obtaining purified
samples of individual
genes facilitate the
study of gene structure
and expression.

3. The main properties of a vector
• Self replication (It should have an Origin of replication ( known as ORI)
• Carry genes that are identifiable by plating or biochemical tests
(Reporter genes and Selectable markers).
• Introduction ability (able to penetrate particular cells).
• Must possess restriction site or Multiple Cloning Site( MCS or
polylinker site) for insertion of DNA molecule

4. Why E.Coli
• Safe
• Short lifetime
• Known genome

5. properties of plasmid
Extra-chromosomal, double
stranded, circular DNA
molecule.
It sizes 1-200 kb.
Can carry a fragment of
DNA up to12 kb.
Found in prokaryotic and
eukaryotic cells.
Replicate as independent
units as the host cell
multiply.
Carry genes (selectable
markers) for heavy metals
tolerance, antibiotic
resistance or able to
metabolize toxic organic
compounds.
Can be easily isolated and
purified from the host cell.

6. Shuttle vectors
• DNA molecules that can replicate in multiple
host organisms (different type of cells or
organisms)
• plasmids contain origin of replication and
selectable marker for more than 1 host
Example: plasmids for the cloning and expression
of genes in the yeast Saccharomyces cerevisiae
contain both replication origins and selectable
markers for both E. coli and yeast
Most DNA
manipulations will be
performed using E.coli
as a host, prior to
transformation of the
final DNA construct into
yeast

7. RNA production plasmids
• They are molecular constructs designed to
facilitate the production of RNA in a host cell.
• the foreign DNA fragments cloned into them
can be transcribed into RNA.
• plasmids contain the promoter sites for an
RNA polymerase
• Example:
1. T3 promoter
2. T7 promoter
3. SP6 promoter
RNA made by this
method is often
used as probes for
hybridization in
Northern blotting.

9. The order of synthesis that distinguishes it
from the other plasmids synthesized in the
same laboratory.
Raymond L. Rodriguez.
Francisco Bolivar Zapata
Plasmid

11. Plasmid
University of
California
Has 3 additional features:
1. High copy number
2. Blue-white Screening
3. multiple cloning site
or polylinker

12. Copy number of
pBR322
Copy number of pUC
High copy number →without
the need for chloramphenicol
amplification.

13. Screening by insertional inactivation of a
resistance gene

14. Replica plating

15. Replica plating

26. Systems have therefore been devised
for the shuttling of DNA fragments
between vectors without having to
use restriction enzymes

27. Gene shuffling between plasmids
using recombination

28. Gene shuffling between plasmids
using recombination

29. Gene shuffling between plasmids
using recombination

30. Gene shuffling between plasmids
using recombination

31. Shortcoming of plasmid
The efficiency at which
the plasmid is
transferred to a
bacterial cell is very
low.
The capacity of
plasmids to carry large
fragments of foreign
DNA is limited