Experiment 2: Gel Electrophoresis of DNA
What is Electrophoresis? Electrophoresis is a technique used in the laboratory that results in the separation of charged molecules. In this CyberLab we are separating molecules of DNA that we got from Restriction Digestion. DNA is a negatively charged molecule, and is moved by electric current through a matrix of agarose. Click on the image to the left to see a larger image of typical equipment used in electrophoresis.
What is Agarose? Click on the image below which you think represents agarose.
You may be wondering what exactly a gel is, and what it has to do with agarose. Let's find out by "making" a gel. Purified agarose is in powdered form, and is insoluble in water (or buffer) at room temperature. But it dissolves in boiling water. When it starts to cool, it undergoes what is known as polymerization. Rather than staying dissolved in the water or coming out of solution, the sugar polymers crosslink with each other, causing the solution to "gel" into a semi-solid matrix much like "Jello" only more firm. The more agarose is dissolved in the boiling water, the firmer the gel will be. While the solution is still hot, we pour it into a mold called a "casting tray" so it will assume the shape we want as it polymerizes (otherwise it will just solidify in the bottom of the flask wasting the expensive agarose). Look through the sequence of images below to learn how to prepare a gel.
Imagine you are a DNA molecule. If you were inside an agarose gel, your environment would resemble a very dense spider web. If you are a small fragment, you could easily crawl through the spaces in between the webs (they are too tough for you to just pull out of the way). But as you increase in length, it gets harder and harder for you to fit through the spaces. If it were a race between you and another DNA molecule, who would win? Do you think the same would hold true for any charged molecule?
Now it's time to take the DNA we digested in Experiment 1 and load it on the gel we just prepared. So again, follow through the pictures below to load and run our gel.
Congratulations! You have now digested a piece of DNA with Restriction Enzymes, separated the digested fragments by Agarose Gel Electrophoresis on a gel you poured, and analyzed and documented your results. And you don't even have to clean up after yourself....
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