Introduction to Gel Electrophorsis

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Introduction to Gel Electrophorsis
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Model of DNADNA is Comprised of Four Base
Pairs
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Deoxyribonucleic Acid (DNA)
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DNA Schematic
O
Phosphate
P
O
O
Base
O
O
CH2
Sugar
O
P
O
O
Phosphate
Base
O
O
CH2
Sugar
OH
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DNA Restriction Enzymes
Evolved by bacteria to protect against viral
DNA infection Endonucleases cleave within
DNA strands Over 3,000 known enzymes
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Enzyme Site Recognition
Restriction site
Palindrome
Each enzyme digests (cuts) DNA at a specific
sequence restriction site Enzymes recognize
4- or 6- base pair, palindromic sequences (eg
GAATTC)
Fragment 2
Fragment 1
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5 vs 3 Prime Overhang
Enzyme cuts
Generates 5 prime overhang
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Common Restriction Enzymes
EcoRI Eschericha coli 5 prime overhang
Pstl Providencia stuartii 3 prime overhang
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The DNA DigestionReaction

• Restriction Buffer provides optimal conditions
• NaCI provides the correct ionic strength
• Tris-HCI provides the proper pH
• Mg2 is an enzyme co-factor

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AgaroseElectrophoresisLoading

• Electrical current carries negatively-charged
  DNA through gel towards positive (red) electrode

Buffer
Dyes
Agarose gel
Power Supply
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AgaroseElectrophoresisRunning

• Agarose gel sieves DNA fragments according to
  size
• Small fragments move farther than large
  fragments

Gel running
Power Supply
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Analysis of Stained Gel

• Determine
• restriction fragment
• sizes
• Create standard curve using DNA marker
• Measure distance traveled by restriction
  fragments
• Determine size of DNA fragments
• Identify the related
• samples

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Molecular Weight Determination
Fingerprinting Standard Curve Semi-log

• Size (bp) Distance (mm)
• 23,000 11.0
• 9,400 13.0
• 6,500 15.0
• 4,400 18.0
• 2,300 23.0
• 2,000 24.0

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Agarose Gel Electrophoresis

• The standard method for separating DNA fragments
  is electrophoresis through agarose gels.

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Agarose Gel Electrophoresis

• The standard method for separating DNA fragments
  is electrophoresis through agarose gels.
• Agarose is a polysaccharide like agar or pectin
  derived from seaweed

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Agarose Gel Electrophoresis

• The standard method for separating DNA fragments
  is electrophoresis through agarose gels.
• Agarose is a polysaccharide like agar or pectin
  derived from seaweed
• It dissolves in boiling water and then gels as it
  cools

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Agarose Gel Electrophoresis

• A comb is placed in the liquid agarose after it
  has been poured
• Removing the comb from the hardened gel produces
  a series of wells used to load the DNA

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Agarose Gel Electrophoresis

• DNA is applied to a slab of gelled agarose

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Agarose Gel Electrophoresis

• DNA is applied to a slab of gelled agarose
• The sample is loaded with a loading
  buffercontaining dyes and glycerol or sugar

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Agarose Gel Electrophoresis

• DNA is applied to a slab of gelled agarose
• The sample is loaded with a loading
  buffercontaining dyes and glycerol or sugar
• Electric current is applied across the gel

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Agarose Gel Electrophoresis

• DNA is negatively charged (due to PO4)

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Agarose Gel Electrophoresis

• DNA is negatively charged (due to PO4)
• Migrates from the negative (black) electrode to
  the positive (red) electrode.

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Agarose Gel Electrophoresis

• Rate of migration of DNA through agarose depends
  on the size of DNA

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Agarose Gel Electrophoresis

• Rate of migration of DNA through agarose depends
  on the size of DNA
• Smaller DNA fragments move more quickly

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Agarose Gel Electrophoresis

• Rate of migration of DNA through agarose depends
  on the size of DNA
• Smaller DNA fragments move more quickly
• Rate of migration is inversely proportional to
  the log10 of molecular weight

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Agarose Gel Electrophoresis
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Agarose Gel Electrophoresis

• Concentration of agarose also affects migration

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Agarose Gel Electrophoresis

• Concentration of agarose also affects migration
• Higher concentration of agarose, the more it
  retards the movement of all DNA fragments

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Agarose Gel Electrophoresis

• Concentration of agarose also affects migration
• Higher concentration of agarose, the more it
  retards the movement of all DNA fragments
• Small DNA fragments require higher concentrations
  of agarose/ Lg fragments low concentrations

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Agarose Gel Electrophoresis

• Agarose gels must be prepared and run in a buffer
  containing ions.

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Agarose Gel Electrophoresis

• Agarose gels must be prepared and run in a buffer
  containing ions.
• Ions are charged particles (like those found in
  salt) and are necessary to carry a charge

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Agarose Gel Electrophoresis

• During electrophoresis water undergoes hydrolysis
  H2O ? H and OH-

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Agarose Gel Electrophoresis

• During electrophoresis water undergoes hydrolysis
  H2O ? H and OH-
• The anode ( /red) pole becomes alkaline because
  OH- will accumulate at this pole
• The cathode (-/black) pole becomes acidic because
  H will accumulate at this pole

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Agarose Gel Electrophoresis

• Buffers prevent the pH from changing by reacting
  with the H or OH- products

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Agarose Gel Electrophoresis

• The buffer is either TBE or TAE
• TBE is made with Tris/Boric Acid/EDTA
• TAE is made with Tris/Acetic Acid/ EDTA

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Agarose Gel Electrophoresis

• The voltage applied to the gel affects how
  quickly the gel runs

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Agarose Gel Electrophoresis

• The voltage applied to the gel affects how
  quickly the gel runs
• The higher the voltage, the more quickly the gel
  runsBut that often reduces the quality of the
  DNA separation

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Agarose Gel Electrophoresis

• The voltage applied to the gel affects how
  quickly the gel runs
• The higher the voltage, the more quickly the gel
  runsBut that often reduces the quality of the
  DNA separation
• gtgtgtgtgtgtgtgtgtgtIt also generates heat which reduces
  the quality of the DNA separation

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Agarose Gel Electrophoresis

• To make DNA fragments visible after
  electrophoresis, the DNA must be stained

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Agarose Gel ElectrophoresisA gel stained with
Methylene blue