Agarose Gel Electrophoresis

If you are looking at this post, you probably need to actually pour, load and run an agarose gel.  First off, agarose is one of the polysaccharides found in agar, a seaweed product that, when melted, has the consistency of stiff Jello.  In fact, not only is agar an ingredient in semisolid culture media for microbes,  it is also a vegetarian alternative to gelatin.

Agarose gel electrophoresis is a simple method for verifying the status and getting a qualitative estimate of the yield of extracted nucleic acids (DNA or RNA).  It can also be used for separating nucleic acids of various sizes, such as DNA following digestion with restriction enzymes.  It can also be the first step in a more involved procedure such as Southern or Northern blot analysis in which the nucleic acids are transferred to a supportive membrane and then hybridized with probes to identify specific fragments (Southern) or specific transcripts (Northern).  We are going to use this method to check our extracted DNAs and to determine the success of our attempts at PCR.

Below you’ll find a description of the specific buffer system and procedure used to prepare and pour a gel.

BUFFERS (Gel and Running)

We’ll be using a “FastGel” buffer system promoted by Promega, Inc.  It allows gel with a single comb to run to completion in about 20 min.  This is because the buffer combination enables the use of higher voltages (200 V)  for a short period of time.  This voltage with the standard 1x/1x buffer system would quickly lead to excessive heat and melting of the gel.

If you need to run a gel and the working stocks are out or low, the following recipes will allow you to make more.

1X TAE (gel buffer)

In the fridge are several small bottles with 50X TAE.  These are stocks that you will use to make either the gel or running buffer.  To make the 1 L of 1X gel buffer, measure 20 ml of 50X TAE and add it to 980 ml of deionized water (in the large container near one of the lab sinks).  Mix well before using.

0.25 X TAE (running buffer)

To make 500 ml of running buffer, add 2.5 ml of 50X TAE to 498 ml (close enough) of deionized water and mix well.  Or, you can add 250 ml of well-mixed 1x TAE to 750 ml of dei water.

THE GEL

Pouring a 1% agarose/1x TAE gel:

1. Obtain a 250 ml Erlenmeyer flask
2. Measure out 1 g agarose and 100 ml of 1X TAE (gel buffer)
3. Pour the agarose and approximately 80 ml of the gel buffer into the flask and swirl
4. Allow the agarose to hydrate for about 5 min. (this allows for more even melting)
5. Place a small Erlenmeyer into that holding the agarose/buffer mix (rattling of the small flask will alert you that the solution is boiling)
6. Microwave the solution at roughly 30 sec intervals until the agarose is completely melted (hold the flask up into the light to verify that all agarose beads have melted).  Use several layers of paper toweling to protect your hand from the heat and be careful when you first grip the flask in case the hot agarose boils over/sprays!
7. Add the remaining 1x TAE to the flask and swirl (this will help the mix cool down faster).
8. Checking every few minutes, let the solution cool until you can stand to hold onto the flask without burning yourself.  At this point, it is cool enough to pour into the gel molds (see the next section for help on setting up the gel mold) without either damaging the mold or burning you.
9. Let the gel cure for at least 20 min.
10. If you are doing this a day early, wrap the gel in the mold in plastic wrap and place in the fridge until you are ready to use it (if the lab supply of combs is running low, remove the comb(s) for other to use).

Setting up a gel mold:

1. Obtain a gel mold and make sure that the end walls are positioned so as to create a four-sided box in which to pour the agarose (make sure the screws are secure – do not tighten too much, the screws are plastic and can break!).  If you are concerned that the mold still might leak, you can carefully add lab tape  around each end of the mold so that the end-wall/mold body is sealed.
2. If you are just running your set of reaction, place a comb (10 or 12 well) in the upper comb slot.  If you and another group wish, you can add a second comb. [you cannot insert combs once the gel has set!]
3. Place the mold with combs on a level surface and pour in your warm agarose mix.

When the gel is ready to load

1. Once the gel is cured, it will have become opaque (cloudy milky in color) and be fairly stiff to the touch.  At this point you can carefully remove the combs.
2. Carefully remove the combs by pulling them slowly straight up.
3. Loosen the screws that hold the end walls in place (Do NOT remove them!).
4. Push then end walls down so that the agarose at the top and bottom is now exposed.  (You can re-tighten the screws to help keep them in the lowered position.)
5. Place the gel on the platform in the gel rig. [Make sure that the wells are closest to the negative (black) electrode so that the DNA can migrate towards the positive (red) electrode.  Unless the gel rigs have been moved, this normally means the wells in the top half of the gel are farther away from you than the other end of the gel.
6. If needed, add enough 0.25 X running buffer to just cover the gel AND to fill the wells.