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Outline
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6.
What Is Cloning?
Using asexual reproduction to obtain organisms that are
genetically identical to one another, and to the parent
Clones produced are only identical genetically; the actual
appearance and behavior of the clones will be influenced by
other factors such as their environment
Example 1:
to propagate plants by taking cuttings, and produce a number
of plants that are identical to the parent. These new plants are
clones
Example 2:
Purifying a bacterial strain by picking a single colony for
inoculation a series of fresh cultures is also a form of cloning
Column purification
Gel electrophoresis
Ethidium bromide agarose gel
mob
gene
Plasmid
mob
protein
nic
site
Plasmid Vectors
Gene cloning requires specialized tools and
techniques
The Vehicles
Transport the gene into the host cell and is
responsible for its replication
Must be capable of entering a host cell, replication
to produce multiple copies of itself
Transformation protocols:
1. Preincubation:
2. Incubation
. DNA is added, and the cell suspension is further
incubated at 0oC. The cations are thought to
neutralize negatively charged phosphates in the
DNA and the cell membrane
3. Heat shock
4. Recovery
If a new fragment of
DNA is ligated into one
of these sites, the
antibiotic-resistance
genes become altered
This is called
insertional
inactivation of the
selectable marker
Identification of recombinants
1. Antibiotic resistance selectable marker
2. lacZ selectable marker
3. Colony hybridization
4. Colony-PCR
End of Lecture 6
Thank You