Gel Electrophoresis Lab

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Gel Electrophoresis Lab
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Agarose Gel Electrophoresis

• Electrolysis the splitting of water using
  electricity
• current splits water into hydrogen ions (H) and
  hydroxyl ions (OH-)
• Electrophoresis a method of separating charged
  molecules in an electrical field DNA has an
  overall negative charge
• Used to separate DNA fragments by size

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Agarose Gel Electrophoresis

• We will be using agarose gel electrophoresis to
  determine the size of DNA fragments made by using
  2 different restriction enzymes on lambda
  DNA-Hind III and EcoRI. We will run lambda DNA
  without restriction enzymes as well.
• 3 vials
• Lambda DNA
• Lambda DNA cut with EcoRI
• Lambda DNA cut with HindIII

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Lambda Phage DNA

• Genomic DNA of a bacterial virus
• Attacks bacteria by inserting its nucleic acid
  into the host bacterial cell
• Replicates rapidly inside host cells until the
  cells burst and release more phages
• Harmless to man and other eukaryotic organisms

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What is needed for restriction digestion?

• Template DNA, uncut DNA, often bacterial phage
  DNA (Lambda DNA)
• Restriction enzyme(s), to cut template DNA
• Restriction Buffer, to provide optimal conditions
  for digestion
• Water Bath

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DNA is negatively charged.
An agarose gel is used to slow the movement of
DNA and separate by size.
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How fast will the DNA migrate?
strength of the electrical field, buffer, density
of agarose gel

• DNA is negatively charge due to the phosphate
  groups. This will
• cause the DNA to migrate to the positive
  electrode
• Migration is based on the size of the DNA
• Small DNA move faster through the agarose gel
  than large DNA
• gel electrophoresis separates DNA according to
  size

small large
Within an agarose gel, linear DNA migrate
inversely proportional to the log10 of their
molecular weight.
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Electrophoresis Equipment
Power supply?
Gel tank?
?Cover
Electrical leads ?
Casting tray?
Gel combs?
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Loading the Gel
Carefully place the pipette tip over a well and
gently expel the sample. The sample should sink
into the well. Be careful not to puncture the
gel with the pipette tip.
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Running the Gel
Place the cover on the electrophoresis chamber,
connecting the electrical leads. Connect the
electrical leads to the power supply. Be sure
the leads are attached correctly - DNA migrates
toward the anode (red). When the power is turned
on, bubbles should form on the electrodes in the
electrophoresis chamber.
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Cathode (-)
DNA (-) ?
? wells
? CarolinaBromophenol Blue
Gel
Anode ()
After the current is applied, make sure the Gel
is running in the correct direction. Bromophenol
blue will run in the same direction as the DNA.
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Actual Results of Restriction Enzyme Digestion

• Lane 1, DNA markers (HindIII lambda digest)
• lane 2, uncut lambda DNA
• lane 3, lambda DNA digested with PstI
• lane 4, lambda DNA digested with EcoRI
• lane 5, lambda DNA digested with HindIII

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DNA Marker Standard Curve

• 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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Applications

• Recombinant DNA Technology
• DNA Cloning
• Constructing DNA Libraries
• Southern Blot Hybridization