DNA 3: Gel Electrophoresis

Aim:  What is gel electrophoresis?

 

Objectives:
Students will be able to…

1) List the steps involved in a gel electrophoresis experiment;

2) Describe the role of restriction enzymes and how they function;

3) Explain how gel electrophoresis separates DNA molecules present in a mixture;

4) Describe the relationship between fragment size and migration rate in a gel;

5) Analyze separate DNA fragments with gel electrophoresis;

6) Explain the impact of gel electrophoresis on today’s society.

Materials:
Gel Electrophoresis chamber, agarose gel, buffer solution, food dyes, pipettes, power source, worksheets, LCD projector, computer, Smart Board.


Motivation:
Visit the Nova website, It Takes a Lickinhttp://www.pbs.org/wgbh/nova/sheppard/lab01.html 
and review the crime scenario outlined.  Walk students through the steps involved in gel electrophoresis.
 
Lesson:
Review the steps in Gel Electrophoresis using the Virtual Gel Electrophoresis website:
http://learn.genetics.utah.edu/content/labs/gel/
 
Activity:
Gel Electrophoresis with food dyes.
  (This activity may be spread over two days.)

Part 1: Prepare a 1% Agarose Gel

Add 0.5g agarose to 50 mL distilled water, heat 1 minute 20 seconds. Let it cool before pouring into the gel plates.  Also be sure the gel plates have been taped securely and contain the well combs prior to pouring.

*this is the protocol for one gel


Part 2 (Day Two):  Prepare the food dyes.
1. While the gel is cooling, prepare the food dye samples.
2. 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.
3. In each test tube place one drop of food dye and three drops of 50% glycerol.  Repeat this for each of the sample.
4. Remove the comb from the gel by gently pulling straight up.
5. When moving the gel, be very careful to hold the slide flat.  If you tilt the slide, the gel can slip off.

 

Part 3 (Day Two): Loading and Developing the Gel
6.
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.
7.
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.
8.
Decide what sample you are going to put in each well and write it down on your worksheet. 
9.
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. 
10.
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.
11.
Repeat this step with a new capillary tube for each of your samples.
12.
After all of the samples are loaded, assemble your battery pyramid and connect the wires to the batteries.
13.
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!
14.
Watch for bubbles to form on the wires in the TBE.  This will tell you that the current is flowing.
15.
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. 
16.
When the gel is done developing, unclip the alligator clips and take the battery pyramid apart.
17.
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.    
18.
Pour the TBE buffer into the sink.

19. Observe the results of the gels.

 

As the electrophoresis demo is running, have students complete the Worksheet, "DNA Fingerprinting: You Be the Judge."

ĉ
Steve Gallagher,
Dec 29, 2009, 8:51 AM
Comments