Bio 181

Downtown Campus Biology > Biology 181 > Lesson 12 Activities > Gel Electrophoresis of Dyes- page 3

Gel Loading Practice

For many students, the hardest step in performing gel electrophoresis is loading the samples into the gel. It takes practice to get it right, and practice is what you're about to do!

In the student refrigerator, locate kit # R-10. Take this kit to your desk. Open the kit and carefully remove one sample loading gel. (It feels like Jello, doesn't it?) You'll notice that one side has wells in it. You can feel these holes.

Place the sample gel into the plastic container from kit # 216. Make sure that you place the gel so that the wells are up - the holes should be facing up.

Add tap water to the plastic container with the sample gel. You want to add just enough water to cover the gel completely. Don't fill the container with water.

Using the micropipettor

Now you'll need to familiarize yourself with a common tool used in biology - the micropipettor. Micropipettors allow you to measure very small quantities of liquid, quantities that are too small to measure in a graduated cylinder or in a glass pipettor like you used in the photosynthesis lab.

	Get kit #197. The micropipettor that you'll use in this activity can measure in from 5 to 50 microliters (ul). How many microliters are in a liter? If you answered 1 million, you're right. To hold the micropipettor, wrap your fingers around the body of the instrument, and set your thumb on the top knob.
	Notice the knob on the top of the micropipettor. Turn this knob to adjust the volume. In this activity, you'll measure 15 ul of each sample. If you need to adjust the volume, please turn the knob slowly and carefully. Micropipettors are precision instruments, and they are not cheap. For the same reason, please handle the micropipettor carefully - avoid dropping it or rough handling.
	Important! Before inserting the micropipettor into any solution, make SURE that you have a disposable yellow tip on the end. The solution will remain in this tip - it shouldn't ever go into the micropipettor itself. To put a tip on the end of the micropipettor - press the micropipettor firmly into the wide end of the yellow tip.
	Pushing on the top knob with your thumb will allow you to "suck up" and "spit out" solutions. First, press your thumb down on the knob. You'll notice there's a natural stop when the knob is about halfway down. We'll call this the "first stop". When you have solution in the micropipettor, pushing the knob to the first stop will eject most, but not all, of the solution.
	With the knob pushed down to the first stop, push a little harder so that the knob goes all the way down. We'll call this the "second stop". When you have solution in the micropipettor, pushing the knob to the second stop will eject the last little bit of solution.
	Look at the blue button on the side, towards the top of the micropipettor. If you push on this, what happens? The yellow tip should come off. Give it a try (but try not to let the tip fall to the floor!)

Loading your sample gel

	Now, you're ready to practice loading the sample gel. Make sure that the yellow tip is firmly attached to the micropipettor. Fill a beaker with tap water.
	Take out the sample loading solution from your kit # R-15. Unscrew the top and place the yellow tip of the micropipettor into the tube. Using your thumb, press the knob halfway, to the first stop. Now, slowly let your thumb up. This will draw the sample solution into the yellow tip. Slowly move the micropipettor tip out along the edge of the sample vial - this should remove any solution that remains stuck to the outside of the yellow tip.
	Now that you have solution in your micropipettor, keep the tip pointing down. If you turn the micropipettor sideways, upside down, etc., solution can move from the tip into the micropipettor itself. This will damage the micropipettor!
	Set the sample vial back in the tray, and put the cap back on it. It is good practice to immediately cap a sample after using it. (Usually you do electrophoresis on small samples of DNA or protein. These samples are often irreplaceable, so you want to carefully guard against any accidental spills.) Make sure you hold the micropipettor vertically - you can't set it down to cap the sample vial!
	Put the tip of the micropipettor into the top of one of the wells in the sample gel. Don't push the tip too far into the well - you may pierce the bottom of the well with the yellow tip. With your thumb, push the knob down to the first stop. This will release most of the sample into the well. Wait at least one full second, then push down to the second stop. This will release the last bit of sample. Keep your thumb on the knob!
	The sample you're loading contains glycerol - this makes it heavier than the water covering the gel. When you release the sample, it should drop down into the well instead of floating away, assuming your tip was placed at the top of the well. Carefully lift the micropipettor out of the water. Once it's out of the water, you can release your thumb. (If you take your thumb off the knob while the micropipettor is still in the water, you'll suck up some of the water.)
	Now wash your yellow micropipettor tip. Place the tip into the beaker of water. Push the knob down with your thumb to the first stop, take in some water, push down to the first stop, then the second stop, to eject the water. Repeat this several times until no sample is visible in the tip. (If you're doing gel electrophoresis on valuable DNA or protein samples, usually you change tips in between each sample to avoid contamination. Since we're only practicing in this activity, we'll rinse the tips to spare the landfill and spare the extra expense of yellow tips.)
	So, if you're like most students, your first attempt at loading a gel wasn't perfect. The most common mistake is that the sample solution floats away, it doesn't drop into the well. This outcome has two likely causes: The tip was too high, it wasn't close enough to the well, when you released the sample The gel is upside down - the openings to the wells aren't face up. Loading a gel takes a steady hand, and practice helps.
	Practice loading another well. Draw 15 ul of sample into the micropipettor tip, cap the sample and set it aside. Insert the tip into the top of the well, push the knob with your thumb to the first stop, then the second stop. With your thumb still pressing the knob down, pull the micropipettor out of the water. Wash the yellow tip in the beaker of water. Practice loading gels until you get at least 2 wells loaded properly.
	Eject the tip and throw it in the garbage. Rinse the practice gel with water. If it isn't falling apart or torn, please return it to kit # R-10 for another student to use. If it is falling apart or torn, please throw the sample gel in the garbage.

15 minutes has probably passed, and your gel is probably set. Now we'll prepare your gel for use!

	If it's not already at your desk, take your casting dish with the gel to your desk. Carefully remove the masking tape from both edges. Hopefully no liquid runs onto the desk!
	Next, remove the comb. Place your index fingers on either side of the comb. Lift up, slowly but firmly. There will be some resistance. Eventually, the gel will unstick and the comb will come out with a little "slurp" sound.
	You should have perfectly formed wells in your gel. You'll be using the middle 6 wells for loading dye samples.

Please get someone at the front desk in the biology lab to come to your desk and look at your gel. They will verify that is has been made properly and give you lab points.

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