Transcript Transformation Lab

Transformation Lab
What are plasmids?
Circular sequences of DNA that can be
incorporated into a bacterial host genome.
What makes them so special??
They contain:
-Origin of Replication
-Selection Marker Gene (Amp)
-Color Marker for easy selection
pGREEN Plasmid
Beta-galactosidase and X-gal
• The beta-galactosidase gene encodes a protein that normally
cleaves the disaccharide sugar lactose into galactose and
glucose. However, the enzyme will also cleave many other
molecules that contain structures resembling the galactoseglucose link. Researchers have developed several chemicals
that, when cleaved by beta-galactosidase, produce colored
products.
One of these color indicators is the chemical 5-bromo-4-chloro3-indolyl-β-D- galactoside. Fortunately, the chemical is usually
referred to by its common name, X-gal.
X-gal is colorless, but when it is cleaved by beta-galactosidase,
one of the products is dark blue. Therefore, if you grow bacteria
that produce beta-galactosidase on media containing X-gal, the
colonies will be bright blue.
pBLU Plasmid
Cloning
• Cloning really means inserting DNA so that it
is propagated in that cell and its progeny.
• In order to do this in bacteria, the “gene of
interest” has to be inserted into the plasmid
(which is used as a vector). This is done in
several steps.
Restriction
Enzymes
•Restriction
enzymes Cut DNA
at specific
sequences.
•DNA Ligase
reattaches that
DNA into the
plasmid