Agarose Gel Electrophoresis

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Revision as of 16:51, 15 October 2007

It is a method used in biochemistry and molecular biology to separate DNA, RNA, or protein molecules by size. This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field (electrophoresis). Applications

   * Estimation of the size of DNA molecules following restriction enzyme digestion, e.g. in restriction mapping of     cloned DNA.
   * Analysis of PCR products, e.g. in molecular genetic diagnosis or genetic fingerprinting
   * Separation of restricted genomic DNA prior to Southern transfer, or of RNA prior to Northern transfer.

The advantages are that the gel is easily poured, does not denature the samples, and is physically firmer than polyacrylamide. The samples can also be recovered.

The disadvantages are that gels can melt during electrophoresis, the buffer can become exhausted, and different forms of genetic material may run in unpredictable forms. Preparation

There are several methods for preparing agarose gels. A common example is shown here. Other methods might differ in the buffering system used, the sample size to be loaded, the total volume of the gel (typically thickness is kept to a constant amount while length and breadth are varied as needed). The vast majority of agarose gels used in modern biochemistry and molecular biology are prepared and run horizontally.[citation needed]

  1. Make a 2% agarose solution in 100ml TAE. A solution of up to 4% can be used if you analyze small DNA molecules, and for large molecules, a solution as low as 0.8% is preferable. Use 15-100 mL, depending on the size of the gel.
  2. Bring the solution to the boil to dissolve the agarose, preferably in a microwave oven.
  3. Let the solution cool down to about 60 °C at room temperature, or water bath. Stir or swirl the solution while cooling.

Wear gloves from here on, ethidium bromide is a mutagen, for more information on safety see ethidium bromide

  1. Add 5 µl ethidium bromide stock per 100 ml gel solution. Be very careful when handling the concentrated stock. Some researchers prefer not to add ethidium bromide to the gel itself, instead soaking the gel in an ethidium bromide solution after running.
  2. Stir the solution to disperse the ethidium bromide, then pour it into the gel rack.
  3. Insert the comb at one side of the gel, about 5-10 mm from the end of the gel.
  4. When the gel has cooled down and become solid, carefully remove the comb. The holes that remain in the gel are the wells or slots.
  5. Put the gel, together with the rack, into a tank with 0.5x TBE. Ethidium bromide at the same concentration can be added to the buffer. Make sure the gel is completely covered with TBE, and that the slots are at the end electrode that will have the negative current.[citation needed]

[edit] Procedure

After the gel has been prepared, use a micropipette to inject about 2.5 µl of stained DNA (a DNA ladder is also highly recommended). Close the lid of the electrophoresis chamber and apply current (typically 100 V for 30 minutes with 15 ml of gel). The colored dye in the DNA ladder and DNA samples acts as a "front wave" that runs faster than the DNA itself. When the "front wave" approaches the end of the gel, the current is stopped. It is now possible to visualize the DNA (stained with ethidium bromide) with ultraviolet light. [citation needed]

Steps: [citation needed]

  1. The agarose gel with three slots/wells (S).
  2. Injection of DNA ladder (molecular weight markers) into the first slot.
  3. DNA ladder injected. Injection of samples into the second and third slot.
  4. A current is applied. The DNA moves toward the positive anode due to the negative charges on its phosphate backbone.
  5. Small DNA strands move fast, large DNA strands move slowly through the gel. The DNA is not normally visible during this process, so the marker dye is added to the DNA to avoid the DNA being run entirely off the gel. The marker dye has a low molecular weight, and migrates faster than the DNA, so as long as the marker has not run past the end of the gel, the DNA will still be in the gel.
  6. Add the color marker dye to the DNA ladder.