Wednesday, April 21, 2010

My Research Explained: Gel Electrophoresis

Today's "My Research Explained" is going to be a double whammy. Last week I decided to talk about two experiments that are crucial in a biology lab. First up is Gel Electrophoresis which has a ton of applications and works on one simple principle. Opposites attract!

I'm referring to electrical charge. Gel electrophoresis is a method of separating particles of varying size. DNA is one of the molecules that is commonly separated in these experiments, but how? First you make a gel:Without going into too much detail you use a chemical called Agarose and dissolve it in water using high temperatures. As the agarose cools it forms a semi-solid matrix of molecules creating a jello-like substance (but don't eat it). If you leave your gel out for too long (a day or so) the water in the gel will evaporate leaving only the agarose matrix. I did this one time and it made a fruit roll-up gel that felt like beef jerky. I had heard that if you add water to this thing, it will re-expand to it's original size. I'll have to try that one day.

Once you have your gel you place it in an electrophoresis box which is just a holder for your gel, some buffer, and has a couple of wires in it. Then you submerge your gel in a buffer (TAE or TBE which stands for the chemicals in each buffer). Now you are ready to load your gel, because in order to separate molecules you need to put molecules in your gel. When you mix your gel you insert a comb while it's still in its aqueous phase. After it hardens you remove the comb and now you have little holes in your gel called wells.

Let's load our gel with DNA. This part takes a little practice because you are trying to get your DNA in a hole about the thickness of your credit card with a tiny tip. You need to take care that you don't rip the gel because even though its semi-solid it is still rather fragile (depending on the percent agarose to water you are using). Carefully pipette your DNA volume into the well and make sure you don't squirt it into the buffer solution. Whatever doesn't get into the well won't be in your gel. In order to make this task a little easier we mix our DNA with a dye that helps with visualization and also adds weight to the DNA so that it sinks in the buffer.

Once your DNA is loaded, you simply plug your gel box in and start it up. You need to make sure that the side of the gel your DNA is on is the same side as the negative terminal of your gel box. This is because DNA just so happens to be negatively charged. This is how we get the DNA moving. When you turn on your power supply you get an electric field across your gel with one side being negatively charged and the other side being positively charged. Since the DNA charge is negative, it will be pushed from this side and attracted to the positive side.

Under normal circumstances all the DNA would move together since the E-field is linear. Since we've loaded the DNA in a gel something special happens. Think of the agarose as a field of tightly packed clumps. If you are small enough you will fit between the clumps with no problem easily making your way through the gel. If you are big you will have a hard time, taking much longer to travel through the gel. When you load DNA of varying length into the gel it experiences this exact phenomenon; longer DNA molecules move much slower than shorter ones. After your experiment is complete you stain your gel with a fluorescent dye so that you can visualize it. You may then see an image like this:
Here I have shown a DNA ladder which is a mixture of molecules of differing lengths. The smallest sized molecules are towards the bottom and the larger molecules are at the top.

And that's all there is to it!

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