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Cutting DNA
Restriction endonucleases
(restriction enzymes)
• sticky ends
• blunt ends
E = genus (Escherichia)
co = species (coli)
R = strain
I = # of enzyme
Blunt & Sticky ends
Pasting DNA
Complementary ends
(sticky ends) H-bond
Ligase forms
phosphodiester bond
to seal strands
A plasmid vector for cloning
1. Contains an origin of replication, allowing
for replication independent of host’s
2. Contains Selective markers: Selection of
cells containing a plasmid
twin antibiotic resistance
blue-white screening
3. Contains a multiple cloning site (MCS)
4. Easy to be isolated from the host cell.
Plasmid vectors
Expression vector
How to clone DNA
Isolation of cloning vector
(bacterial plasmid) & genesource DNA (gene of interest)
Insertion of gene-source DNA
into the cloning vector using
the same restriction enzyme;
bind the fragmented DNA with
DNA ligase
Introduction of cloning vector
into cells (transformation by
bacterial cells)
Cloning of cells (and foreign
Identification of cell clones
carrying the gene of interest
Screening of the clone
The medium in this petri
dish contains the antibiotic
The bacteria on the right
contain Kanr, a plasmid
that is resistant to
Kanamycin, while the one on
the left has no resistance
Note the difference in
Blue/White Color Screening
functional enzyme
nonfunctional enzyme
Selecting Colonies with
Recombinant Plasmids