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Orientation:

The purpose of this activity is to introduce the principles and terminology of electrophoresis and demonstrate the separation of food color dyes with agarose gel electrophoresis.

Supplies:

Electrophoresis

Chamber

1 plastic dish

1 slide box lid or bottom

Aquarium sealant

Large needle

Seizing wire

Hot glue gun and glue         

 

Power Supply

5 nine-volt batteries

1 nine-volt battery clip

2 alligator clips (1 red / 1 black)

Scissors or wire cutters

 

Agarose Gel

Transfer pipettes

2 x 3 glass slide

Well comb (template and Styrofoam)

Tris-Borate-EDTA Buffer (TBE)

Agarose

Graduated cylinder

Erlenmeyer flask

Balance

Microwave or hot water bath

Sample Preparation

 

Small vials or tubes

Tube rack

Permanent marker

Capillary tubes with bulb

Food colors

50% glycerol solution

Access the Student Journal for Activity 7

Access the Student Presentation for Activity 7

Activity 7 - Edible Colors

Building the Electrophoresis Chamber

 

  1. Place the slide box in the center of the plastic dish and trace around the edge with the permanent marker.

 

  1. Extend the lines of the long sides of the slide dish all the way up one side of the plastic dish.  Use the permanent marker to place a spot near the rim of the plastic dish, about 1 cm out from each of the lines. 

 

  1. Carefully use the large needle to punch a hole through the plastic dish at each spot. 

 

  1. Use the scissors or wire cutters to cut two pieces of seizing wire about 10 cm longer than the length of the plastic dish. 

 

  1. Take one piece of wire and make a 90˚ bend 1 cm from the end.  Be careful because the ends of the wire will be sharp.

 

  1. Go down the wire and make another 90˚ bend so that the wire will fit down into the plastic dish and touch both sides. 

 

  1. Position the wire 1 cm above the bottom of the plastic dish (the width of your finger) and make a 90˚ bend that will allow the wire to come out of the hole. 

 

  1. Feed the end of the wire through the hole and bend it into a loop.  Cut off and dispose of any excess wire.
  2. Repeat these steps with the second piece of wire and the second hole.

 

  1. Place the slide box in the center of the plastic dish to make sure that neither of the wires touches it.  Make necessary adjustment to prevent the wires from touching the slide box.  Remove the slide box.

 

  1. Plug in the hot glue gun and allow it to heat up.  Be careful when using the hot glue gun because the gun and the glue will both get very hot.

 

  1. Use the hot glue to secure the wire at each of the 90˚ bends.  Allow the glue to dry.

 

  1. Put a thick bead of aquarium sealant around the rim of the slide box.  Place the slide box open side down into the center of the plastic dish where you traced it.  If necessary add more sealant around the edges to make sure there will be no leaks.  The aquarium sealant will need to dry overnight. 

 

Building the Power Supply

 

  1. Cut the battery clip in half with the scissors, be careful to not cut the wires. 

 

  1. Using the scissors, strip away about 1.5 cm of plastic covering off the wire attached to each half of the battery clip. 

 

  1. Pull the plastic cover off one arm of each of the alligator clips.  Slide the plastic cover onto the wire.

 

  1. Wrap the wire from the negative end of the battery clip (red wire) around the arm of red alligator clip. 

 

  1. Slide the plastic cover back onto the alligator clip to cover the connection. 

 

  1. Repeat these steps for the black wire and black alligator clips.

 

  1. Build a pyramid with the batteries, three on bottom and two on top.  Connect the negative ends of the batteries to the positive ends of the opposing batteries.

 

  1. When you are ready to use the power source attach the red wire to the empty positive end of the battery and the black wire to the empty negative end of the battery.

 

  1. Do not touch the alligator clips on the end of the power supply or the buffer when the power supply is attached to the electrophoresis chamber because it will electrocute you.  Store power supply with the batteries taken apart. 

 

 

 

 

 

 

Preparing the Gel and the Samples

 

  1. For this experiment a 1% Agarose gel was used.  Only a few milliliters of agarose gel solution are needed per gel.  Here is the recipe for 10 ml: 

 

    1. Weigh 0.1 g of agarose powder on the balance
    2. Place it in the Erlenmeyer flask
    3. Add 10 ml of 1X TBE

 

  1. The agarose and TBE will need to be heated in order for the agarose to dissolve.  It takes about 1 minute in the microwave, or you can place the flask in a hot water bath.  When the agarose is melted, the solution will be clear and there will be no particles floating in it.  Caution: the flask and the liquid will be very hot!  Handle with care.

 

  1. Allow the agarose solution to cool.  It will be ready when the bottom of the flask is cool enough to touch without burning your skin. 

 

  1. While the agarose solution cools, take the glass slide and lay it on a hard, level surface. 

 

  1. Take the two binder clips and the well comb.  Grip the well comb in the two binder clips so that the comb is level and just above the glass slide.

 

  1. Take a transfer pipette and fill it with the agarose solution.  Starting at the comb and moving back, lay the agarose solution on the slide.  Be sure to: work quickly; do not allow any gaps; do not over fill the slide because the agarose will spill over the edge; and pop any air bubbles immediately.

 

  1. Allow the gel to cool and solidify.  The gel will turn opaque when it is set.  (About 5 minutes) 

 

  1. While the gel is cooling, prepare the samples.

 

  1. Label the test tubes/vials with the color of food dye that will be put in them.  Label one for red, blue, green, and yellow.

 

  1. In each test tube place one drop of food dye and three drops of 50% glycerol.  Repeat this for each of the samples.

 

  1. Remove the comb from the gel by gently pulling straight up. 

 

  1. When moving the gel, be very careful to hold the slide flat.  If you tilt the slide, the gel can slip off.

 

Loading and Developing the Gel

 

  1. Place the gel on top of the slide box in the electrophoresis chamber.  Make sure that the slide is positioned so that the row of wells is parallel with the wires.

 

 

  1. Pour enough TBE buffer into the electrophoresis chamber so that it covers the gel.  Do not pour the TBE directly onto the gel; pour it to the side of the gel in the plastic dish.  If the TBE is poured directly on the gel, it could push the gel off of the slide.

 

  1. Decide what sample you are going to put in each well and write it down on your worksheet. 

 

  1. Gently put the bulb on a capillary tube.  Be very careful when handling the capillary tubes because they are very thin glass tubes and they break easily.  To fill the capillary tube, place the tip of it in the sample and it will automatically draw the sample up.  Be careful to keep the sample in the capillary tube and do not let it into the bulb.  Once the tube is almost full you will need to cover the hole on top of the bulb and squeeze the bulb just a little to keep the sample from coming up into the bulb.  Hold the pressure steady so that you do not push the sample out of the tube.  This may take a little practice. 

 

  1. Place the tip of the capillary tube into the buffer right above the well you are going to fill.  Do not put the capillary tube into the well because, if it is bumped, it could punch through the well leaving a hole that would leak out the sample.  Slowly squeeze the bulb more to push the sample out of the capillary tube.  The sample is heavier than water (because you added the glycerol), so it will go straight down into the well.  Some of the sample may come out of the well and get into the buffer. That is all right as long as most of it is in the well.  Dispose of the capillary tube in a container designated for glass disposal.

 

  1. Repeat this step with a new capillary tube for each of your samples.

 

  1. After all of the samples are loaded, assemble your battery pyramid and connect the wires to the batteries.

 

  1. Clip the black alligator clip to the loop on the wire that is behind the wells and the red alligator clip to the loop on the wire that is in front of wells.  Do not touch the buffer while the clips are attached!  You will be electrocuted!

 

  1. Watch for bubbles to form on the wires in the TBE.  This will tell you that the current is flowing.

 

  1. Allow the gel to develop until you can see the colors separate.  The food dyes in the food colors will travel through the gel with the electrical current towards the red wire. 

 

  1. When the gel is done developing, unclip the alligator clips and take the battery pyramid apart.

 

  1. Remove your slide from the electrophoresis chamber and place it onto a white paper towel or piece of paper so you can see the colors better.    

 

  1. Pour the TBE buffer into the sink.

 

  1. Observe the results of the gels.

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