Transcript Library construction - Center for Bioinformatics and

Cutting DNA
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Restriction endonucleases
(restriction enzymes)
• sticky ends
• blunt ends
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Nomenclature
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EcoRI
E = genus (Escherichia)
co = species (coli)
R = strain
I = # of enzyme
Blunt & Sticky ends
Pasting DNA
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Complementary ends
(sticky ends) H-bond
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Ligase forms
phosphodiester bond
to seal strands
together.
A plasmid vector for cloning
1. Contains an origin of replication, allowing
for replication independent of host’s
genome.
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
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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
genes)
Identification of cell clones
carrying the gene of interest
Screening of the clone
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The medium in this petri
dish contains the antibiotic
Kanamycin
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
growth
Blue/White Color Screening
lacZ
lacZ
functional enzyme
X-gal
product
insert
nonfunctional enzyme
X-gal
product
Selecting Colonies with
Recombinant Plasmids