Transformation Lab

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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

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pGREEN Plasmid
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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
  galactose-glucose 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-chloro-3-indolyl-ß-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.
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pBLU Plasmid
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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.

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Restriction Enzymes

• Restriction enzymes Cut DNA at specific
  sequences.
• DNA Ligase reattaches that DNA into the plasmid

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