(thing) by Metacognizant Wed May 22 2002 at 18:47:34

What it is:

An agarose gel is effectively a rectangular filter made of toxic Jello.

Why you'd want to do it:

You use an agarose gel for gel electrophoresis, which is a method for sorting and visualizing DNA, RNA, or protein molecules by mass, as measured in base pairs (for DNA or RNA) or in amino acids (for proteins). This is useful for
  • Testing the success of a DNA or RNA extraction
  • Ascertaining the presence of multiple alleles for protein production in members of a population
  • Choosing DNA samples that are promising candidates for sequencing (larger fragments = more promising)

What you'd do it on:

When using a gel, you can run
  • The product of a PCR (DNA/RNA amplifying) reaction
  • The product of an enzymatic, DNA/RNA cutting reaction
  • The product of a minicolumn, DNA/RNA purifying reaction

    ...Among other things.

...And HOW:

The following protocol is what I use to produce a 50 ml, 1% agarose gel. To increase the % agarose (some gels call for as much as 2%), or the volume, just maintain the following proportions.
  1. Set up a gel apparatus. This is made of hard plastic and consists of an electrophoresis chamber with two wells on the side, a removable gel tray, a comb with as many teeth as you have samples/ ladders, and a lid connected to power cords. Place the gel tray in the electrophoresis chamber so that the rubber gaskets on the tray engage the sides of the chamber. You may have to wet the gaskets with water so that they slide in.

  2. Then place the comb at one end of the gel tray- there will be 2 notches that it fits into.

  3. Weigh out .5 g of agarose, a white powder, and dump it into a 250 ml Erlenmyer flask.

  4. Add 50 ml of 1x TAE buffer to the flask. (belgand says S/he uses 40 ml of TBE instead.)

  5. Drop a magnetic stir bar into the flask and cover the top of the flask with a small beaker, to minimize evaporation.

  6. Heat the flask in the microwave for about a minute, or until it boils.

  7. Remove the flask from the microwave and place it onto a magnetic stir plate. Let it stir until it is warm, but not hot. Don't stir it so fast that it forms bubbles, or so slowly that it begins to solidify.

  8. When the mixture is warm, add 2.5 µl of .25 mg/ml concentration Ethidium Bromide solution. Be careful with this- it's a mutagen and suspected carcinogen, so don't let it spill or get on your hands.

  9. Let the mixture stir for another few seconds, then pour it into the gel chamber.

  10. Then leave it for at least 30 minutes, up to several hours, to solidify.

  11. Once the gel is solid, remove the gel tray from the electrophoresis chamber, rotate it 90 degrees, and put it back in.

  12. Remove the comb from the solid gel. There will be a number of small indentations in the gel where the comb made an impression.

  13. For a 9.5 microliter load, mix 5 µl of DNA, RNA or protein sample with 4 µl of ultrapure water and .5 µl of blue dye. (belgand omits the water.)

  14. Then carefully pipette one 9.5 µl load of sample into each indentation, recording which sample corresponds with which lane.

  15. Into one lane alongside those containing your samples, load 5 or 10 µl of a commercially available "ladder", which contains DNA/RNA/protein fragments of known size.

  16. Pour 1x TAE buffer into the two wells of the electrophoresis chamber until they are half full.

  17. Put about 2 µl of .25 mg/ml concentration Ethidium Bromide solution into each well. This will counteract the positive to negative motion of the Ethidium Bromide previously added to the gel, so that it can bind to the DNA/RNA/protein molecules.

  18. Pour in more 1x TAE buffer, until both wells are full and the gel itself is covered to a depth of one or two millimeters.

  19. Cover the electrophoresis chamber with the plastic cover, and plug in the two power chords so that the black, negative one is next to the indentations that you have loaded the samples into, and the red, positive one is opposite them.

  20. Plug the other end of each cord into a power supply, and turn the power supply on at about 50 or 60 volts.

  21. Leave the gel running until the fastest blue dye has moved about 2/3 of the way through the gel. This wil take about 45 minutes to an hour.

  22. When the electrophoresis is finished, turn off the power supply and remove the cover.

  23. Remove the gel, place it under a UV lamp, and photograph.

How it works:

When the DNA/RNA/protein fragments are subjected to the electric current, they migrate towards the positive terminal of the gel apparatus. Small fragments migrate more rapidly than large ones, so by comparing the sample fragments to the fragments of known size present in the ladder, one can estimate the size of the fragments in the sample. The blue dye is used to confirm that the gel is running succesfully, but what is really crucial for visualizing fragments is the Ethidium Bromide. This molecule binds to the fragments, and floresces when exposed to UV light. A photograph taken of the completed gel under UV light will show the sizes of the sample fragments, as well of of the fragments in the ladder, so that the former can be sized with considerable accuracy.
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