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GEL ELECTROPHORESIS VIRTUAL LABORATORY

Welcome to the gel electrophoresis virtual laboratory! Have you ever wondered how scientists work with tiny molecules that they can't even see with the naked eye? Here's your chance to try it yourself. Sort and measure DNA strands by running your own gel electrophoresis experiment. You are holding a small plastic tube with some clear liquid in it. You've been told that the liquid contains DNA strands of several different lengths. Your job is to find out what those lengths are. How will you do it? Let's find out! If the DNA strands were as big as your shoelaces, you could sort them by hand into groups and then measure them. But DNA strands are molecules so tiny that you can't see them even under most microscopes. Is there a way to sort and measure the DNA strands in your tube even though you can't see or touch them? There is! It's called gel electrophoresis. Scientists use gel electrophoresis whenever they need to sort DNA strands according to length. This technique is also useful for separating other types of molecules, like proteins. How does this work? The gel is a filter that sorts the DNA strands. It's like a sponge made of Jell-O with many small holes in it. We place DNA samples into the holes at one end of the gel. Electrophoresis is how we push the DNA strands through the filter. By adding an electrical current, we can make the DNA move. Short strands move through the holes in the gel more quickly than long strands do. Over time, the shorter strands in the sample will move further away from the starting point, versus the longer strands. DNA strands of the same length, will move at the same speed and will end up grouped together. In this way, the DNA strands in the sample sort themselves. Staining the sorted groups of DNA, makes them vis ible to the naked eye. Although we can't see a single strand of DNA, we CAN see large groups of stained DNA strands. These groups show up as bands on the gel.

GEL ELECTROPHORESIS VIRTUAL LABORATORY Now, it's your turn to run a gel. You will run your own gel electrophoresis by following these steps: • Step one - Make the gel. • Step 2 - Set up the gel apparatus. • Step 3 - Load the DNA sample into the gel. • Step 4 - Hook up the electrical current and run the gel. • Step 5 - Stain the gel and then analyze the results. Here is what you will need to make the gel: • powdered agarose • buffer • a flask • a microwave • the gel mold • the gel comb. Making an electrophoresis gel is a lot like making Jell-O, but you don't want to eat this type of gel. We'll begin by placing a small amount of agarose into the flask. Agarose is a dried powder similar to gelatin, but it's made from seaweed. Now, we'll add some liquid buffer to the flask. The buffer is a salt water solution that will let the electrical charges flow through the gel. We've loosely placed plastic wrap over the top of the flask to prevent the liquid from boiling over.

We can now open the microwave by pressing the OPEN button, and place the flask containing the buffer and agarose mixture inside the microwave. We will heat the mixture until the agarose melts into the buffer. We've removed the plastic wrap from the top of the flask.

GEL ELECTROPHORESIS VIRTUAL LABORATORY Now we can pour the melted agarose mixture into our mold. Notice that the mold has tape on each end to hold the melted agarose. Now, we'll place the comb into the gel on one end. The notches in the gel mold hold it in place. Let the gel cool and solidify. This usually takes about half an hour. As the gel cools, tiny holes form in it. Now that the gel has cooled, we'll carefully remove the comb, leaving empty wel ls that will be used for the DNA samples.

Now it's time to set up the electrophoresis box. You will need the gel that you just made, an electrophoresis box, and another bottle of buffer.

GEL ELECTROPHORESIS VIRTUAL LABORATORY

We pour the buffer into the electrophoresis box, then place the gel (still in the mold) into the electrophoresis box. We have removed the tape from the ends of the gel mold. The gel should be just barely submerged in the buffer.

The buffer conducts the electrical current from one end of the gel to the other. It will also keep the gel from drying out during the experiment.

GEL ELECTROPHORESIS VIRTUAL LABORATORY You're ready to load the DNA sample into the gel. Here's what you'll need: Loading buffer, the tube of DNA, the DNA size standard, a micropipettor, the electrophoresis box (containing buffer and the gel), and pipette tips. With a clean pipette tip, use the micropipettor to suck up some of the loading buffer and then add it to the DNA sample.

DNA samples are prepared in a clear liquid solution that would be hard to see if you tried to load it directly into a well. The loading buffer contains a die that makes the sample easy to see. It is also slightly goopy. This makes the DNA sample thicker, so that it will drop into the well, instead of just floating away. The DNA size standard already contains the loading buffer. Next, you will use the micropipettor to transfer the DNA sample from the tube into the well of the gel. First, suck up some of the DNA sample into the pipette tip. Now, we will eject the DNA sample from the pipette into the first well of the gel. In real life, loading the sample into a well takes some practice, so don't be disappointed if you missed your target the first few times.

Using a clean pipette tip, use the micropipettor to suck up some DNA size standard. Transfer the DNA size standard into the next empty well. The DNA size standard contains DNA strands of KNOWN length. Running it on the gel will give you a reference by which to estimate the lengths of the DNA strands in your sample.

GEL ELECTROPHORESIS VIRTUAL LABORATORY

It's time to turn on the electricity and run the gel. When you turn on the power, the black end will generate a negative charge. The red end will generate a positive charge. Together they will pass the current through the gel. DNA has a negative charge. To move the DNA through the gel, you must put the black cord, which is the negative charge, closest to the wells. Plug the black cord from the electrophoresis box into the matching outlet on the power supply. Now, plug in the red cord. Now we can turn on the power supply. Your gel is off and running. Let's look into the gel box to see what is happening. Check for tiny air bubbles coming out of the electrodes at both ends of the electrophoresis box. The bubbles are your proof that a current is running.

Repelled by the negative charge, the DNA moves through the gel towards the positive charge at the other end. Short DNA strands move through the holes in the gel more quickly than long strands do. Over time, the shorter DNA strands will migrate further from the starting point. We can't actually see the migrating DNA bands, but we can see the blue dye from the loading buffer as it migrates.

GEL ELECTROPHORESIS VIRTUAL LABORATORY

You have finished running your gel. We've taken the gel mold with the gel in it out of the electrophoresis box. The time has come for you to analyze your sample, and estimate the length of DNA strands. First, you'll need to stain the DNA in your gel using a DNA staining solution. The stain is a chemical called ethidium bromide, which binds to DNA and shows up under a fluorescent light. Although we can't see any single DNA strands, we can see large groups of stained DNA strands. These groups will show up as bands in the gel. Now, we can drag the gel out of the mold and put it into the DNA staining solution. Because ethidium bromide binds to DNA, it can damage the DNA in your cells. If you stain a gel in real life, be sure to wear gloves and avoid direct contact with the staining solution. It takes about half an hour to stain the DNA in the gel. Once the staining is complete, we remove the gel from the staining solution, and place it on a UV or ultraviolet light box. Now, we can turn our UV box on. The ultraviolet light or UV light from the box can damage your eyes. If you do this in real life, be sure to wear protective goggles. Now you can determine the approximate lengths of the DNA strands in your sample. Compare the bands from the DNA sample with the bands of KNOWN length from the DNA size standard. Electrophoresis cannot tell us the exact DNA lengths of DNA strands, just a good estimate. So, give it your best guess based on the DNA size standards.

GEL ELECTROPHORESIS VIRTUAL LABORATORY The first band in our DNA sample is just about the same as the band here in the DNA size standard which we know is 6000 base pairs. So, we can estimate the size of this band to be six thousand base pairs. The 2nd band in our DNA sample is between three thousand and four thousand base pairs. It seems to be not exactly halfway between them, but maybe slightly closer to three thousand. Remember, these are estimations, so they don't have to be exact. We can estimate about 3,300 base pairs for this band in the sample. The 3rd band in our sample is just about halfway between 1000 and 2000 base pairs. We can estimate that this is around 1500 base pairs.

Congratulations! You have just run your own DNA gel electrophoresis experiment! Now you are ready to answer the questions in your virtual laboratory write-up....