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Gene Technology

Basic steps in DNA extraction

Lysis:
• Detergents
• Organic solvent
• Proteases
(lysozyme)
• Heat

“cell extract”

Genomic DNA prep: removing proteins and
RNA

chloroform

Need to mix gently! (to avoid shearing breakage of the
genomic DNA)
Add the enzyme RNase A to degrade RNA in the aqueous
layer

Precipitation of DNA
Precipitate out of DNA with isoproproanol

Agarose gel electrophoresis
• Agarose Gel Electrophoresis: separate and visualize DNA
fragments based on size
• Agarose is isolated from seaweed and when melted in a
buffer solution and poured into a horizontal tray and as it
cools it will form a semisolid gel containing small pores
through which DNA will travel
• The percentage of agarose used to make the gel
determines the ability of the gel to separate DNA
fragments of different sizes
• (gel % range from 0
...
5%) resolves larger size fragments

Agarose gel electrophoresis
• Agarose Gel Electrophoresis
– To run a gel, it is submerged in a buffer solution that
conducts electricity
– DNA is loaded into small depressions called wells at
the top of the gel
– Electric current is applied through electrodes at
opposite ends of the gel
• DNA migrates according to its charge and size
• Rate of migration through the gel depends on the size of the
DNA because the sugar phosphate backbone makes it
always negatively charged
• DNA migrates toward positive pole and is repelled by
negative pole

Agarose gel electrophoresis
• Agarose Gel Electrophoresis
• Migration distance is inversely proportional to size
of DNA fragment
– Large fragments migrate slowly; smaller fragments
migrate faster
– Tracking dye is added to the samples to monitor DNA
migration during electrophoresis
– DNA can be visualized after electrophoresis by the
addition of DNA staining dyes
• Ethidium bromide: intercalate between DNA base pairs and it
fluoresces under ultraviolet light
• Then a picture can be taken to document the gel results

Agarose gel electrophoresis

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Introduction to Recombinant
Technology and DNA Cloning

DNA

• Restriction Enzymes
Primarily found in bacteria (they use these for defense)
– Cut DNA by cleaving the phosphodiester bond that joins
adjacent nucleotides in a DNA strand
– There are 4 or 6 bp cutters because they recognize
restriction sites with a sequence of 4 or 6 nucleotides

Recognition site – specific base sequence on DNA where
a restriction enzyme binds
...

example: RACECAR or GAATTC
CTTAAG

• Each restriction enzyme has its own unique recognition site
...

Introduction to
Technology
and

Recombinant DNA
DNA
Cloning

• Restriction enzymes
a
...
Some cut DNA to generate fragments with
double-stranded ends called "blunt" ends

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Introduction to Recombinant DNA
Technology and DNA Cloning
• Plasmid DNA – small circular pieces of DNA
found primarily in bacteria
• Are considered extrachromosomal DNA because
they are in the cytoplasm in addition to the
bacteria chromosome
• Are small approximately 1 to 4 kb
• Can replicate independently of chromo

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