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  Section: Biotechnology Methods » Electrophoresis
 
 
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Agarose Gel Electrophoresis

 
     
 
Introduction
Electrophoresis through agarose or polyacrylamide gels is the standard method used to separate, identify, and purify DNA fragments. The technique is simple, rapid to perform, and capable of resolving fragments of DNA that cannot be separated adequately by other procedures, such as density gradient centrifugation. Furthermore, the location of DNA within the gel can be determined directly by staining with low concentrations of the fluorescent intercalating dye ethidium bromide; bands containing as little as 1–10 mg of DNA can be detected by direct examination of the gel in ultraviolet light. If necessary, these bands of DNA can be recovered from the gel and used for a variety of cloning purposes.

This method, whereby charged molecules in solution, chiefly proteins and nucleic acids, migrate in response to electric field is called electrophoresis. Their rate of migration, or mobility, through the electric field depends on the strength of the field, on the net charge, size, and shape of the molecules, and also on the ionic strength, viscosity, and temperature of the medium in which the molecules are moving.
 


  Movement of the DNA in the gel depends on its molecular weight, conformation, and concentration of the agarose, voltage applied, and strength of the electrophoresis buffer.

Materials
  • Submarine gel apparatus, including glass plate, comb, and surround
  • Ethidium bromide: 10 mg/mL
  • Agarose TAE buffer: 0.04 M tris-acetate, 0.001 M EDTA, pH 8.0
  • Ethanol 6 X gel-loading buffer

Preparation and Examination of Agarose Gels

Seal the edges of the clean, dry, glass plate (or the open ends of the plastic tray supplied with the electrophoresis apparatus) with autoclave tape so as to form a mold. Set the mold on the horizontal section of the bench (check with a level).
  1. Prepare a sufficient electrophoresis buffer (usually 1 X TAE or 0.5 X TBE) to fill the electrophoresis tank and prepare the gel. Add the correct amount of powdered agarose to a measured quantity of electrophoresis buffer in an Erlenmeyer flask or a glass bottle with a loose-fitting cap. The buffer should not occupy more than 50% of the volume of the flask or bottle.
  2. Heat the slurry in a boiling-water bath or a microwave oven until the agarose dissolves.
  3. Cool the solution to 60°C and, if desired, add Ethidium bromide (from a stock solution of 1.0 mg/mL in water) to a final concentration of 0.5 mg/mL and mix thoroughly.
    (a) Treat the solution with 100 mg of powdered activated charcoal for each 100-mL solution.
    (b) Store the solution for 1 hour at room temperature, with intermittent shaking.
    (c) Filter the solution through a Whatman No.1 filter and discard the filtrate.
    (d) Seal the filter and activated charcoal in a plastic bag and dispose of the bag in a safe place.


AGAROSE GEL ELECTROPHORESIS


Materials
  • Agarose solution in TBE or TAE (generally 0.7%–1%)
  • 1X TBE or TAE (same buffer as in agarose)
  • Gel-loading dye

Procedure

  1. To prepare 100 mL of a 0.8% agarose solution, measure 0.8 g of agarose into a glass beaker or flask and add 100 mL of 1 X TBE or TAE and 10 mg/mL ethidium bromide.
  2. Stir on a hot plate until the agarose is dissolved and the solution is clear. Allow solution to cool to about 55°C before pouring. (Ethidium bromide)
  3. Add the solution to a concentration of 0.5 mg/mL.
  4. Prepare gel tray by sealing the ends with tape or another custom-made dam.
  5. Place the comb in the gel tray about 1 inch from one end of the tray and position the comb vertically, so that the teeth are about 1–2 mm above the surface of the tray.
  6. Pour 50°C gel solution into the tray to a depth of about 5 mm. Allow the gel to solidify for about 20 minutes at room temperature.
  7. To run, gently remove the comb, place the tray in an electrophoresis chamber, and cover (just until wells are submerged) with electrophoresis buffer (the same buffer used to prepare the agarose).
  8. Excess agarose can be stored at room temperature and remelted in a microwave.
  9. To prepare samples for electrophoresis, add 1 µL of 6X gel loading dye for every 5 mL of DNA solution. Mix well. Load 5–12 µL of DNA per well (for minigel).
  10. Electrophoresis at 50–150 volts, until dye markers have migrated an appropriate distance, depending on the size of the DNA to be visualized. If the gel was not stained with ethidium during the run, stain the gel in 0.5 mg/mL ethidium bromide until the DNA has taken up the dye and is visible under shortwave UV light.


















































































 
     
 
 
     




     
 
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