Molecular Rainbow: Electrophoresis to Identify the
Molecules in a Mixture
Student Materials
Introduction........................................................................................................... 2
Lab Protocol.......................................................................................................... 3
Data Collection Worksheet .................................................................................. 4
Pre-Lab Questions ................................................................................................ 5
Post-Lab Questions and Analysis ....................................................................... 6
Students
You should read the Introduction and Lab Protocol and then answer the Pre-Lab Questions.
Also be sure to answer the questions that are embedded in the Introduction. Completion of the
questions will help you understand the concepts and procedures of the lab. Once you have
completed the lab, complete the Post-Lab Questions and Analysis.
Molecular Rainbow: Electrophoresis to Identify the
Molecules in a Mixture
Introduction
Introduction:
Take just a few seconds and answer this question for yourself: Biology is the study of living things, but
how are living things studied? You are going venture into the field of molecular biology by exploring one
tool that allows the study of biology at the molecular level.
Molecules like DNA, RNA and proteins are not living, but understanding these molecules informs us
about living things. How can you study molecules of DNA, RNA or protein when they are too small to
be seen, even with a microscope? A good first step may be to determine the size of a molecule.
A common way to measure molecular size is by gel electrophoresis, which is a big name for a simple
concept. Here is an analogy to explain gel electrophoresis. Imagine a very dense forest of trees where
each tree is only one foot away from every other tree. You and a mouse must each run through the
forest from point A to point B. You and the mouse begin at the same time.
Which will reach the other side first, you or the mouse? Write your ideas here: __________
__________________________________________________________________________
The gel in gel electrophoresis is like the forest except instead of trees, it is a mesh formed of polymers.
It is a lot like JELL-O, but for electrophoresis the gels are made of agarose, which is purified from
seaweed whereas JELL-O is mostly gelatin made from animal products like collagen. Once solidified,
the gel is submerged in a tank filled with a buffer (a salt solution such as tris-acetate that controls the
pH) that conducts electricity. The molecules to be studied are suspended in the gel, and an electric field
is applied.
In what direction will molecules with a negative surface charge move? __________________
In what direction will molecules with a positive surface charge move? __________________
Molecules with a positive surface charge will move through the gel towards the cathode, the negatively
charged pole. Molecules with a negative surface charge will move through the gel towards the anode,
the positively charged pole. If the relative charges of the molecules are the same, then the molecules
will move through the gel at different rates based on their size, with smaller fragments or molecules
moving faster.
Gel Electrophoresis Unit
Dyes separated by gel
electrophoresis
In this activity, you will use agarose gel electrophoresis to determine what mixture of dyes is found in
two mystery samples.
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Molecular Rainbow: Electrophoresis to Identify the
Molecules in a Mixture
Lab Protocol
Materials:
 Electrophoresis gel box, tray, comb and electrodes
 Power Supply
 p20 Micropipettor and tips
 Microcentrifuge tube rack
 50 mL Graduated cylinder
 500 mL Graduated cylinder
 250 mL Erlenmeyer flask
 500 mL Erlenmeyer flask
 Ruler
 permanent marker
 Agarose
 10X tris-acetate buffer
 6 Standard Dye samples
 2 Unknown samples
 dH2O
Procedure:
Prepare 500 ml 1X TAE buffer:
1. Measure 50 mL of 10X TAE (tris-acetate) buffer using the 50 mL graduated cylinder.
2. Measure 450 mL dH2O using the 500 mL graduated cylinder.
3. Add the dH2O and 10X TAE to a 500 mL Erlenmeyer flask, mix by swirling and label “1X TAE”
If you want to make 20 ml of 1X TAE buffer, you would mix ______ ml of 10X TAE with ______ ml water.
If you want to make 120 ml of 1X TAE buffer, you would mix ______ ml of 50X TAE with ______ ml water.
Prepare a 0.8% agarose gel:
4. Set up the gel tray with comb.
5. Measure 50 mL of 1X TAE using the 50 mL graduated cylinder and place it in a 250 mL Erlenmeyer flask.
6. Go to a weighing station and weigh out 0.4 g of agarose.
7. Add the 0.4 g agarose to the flask containing 50 mL of 1X tris-acetate. This next step should be done with
your instructor’s supervision. Heat the agarose solution in a microwave until the solution is clear. The solution
can boil over easily! In order to prevent spilling, heat the agarose solution in 30-second intervals and mix by
swirling the flask after each interval until the solution is clear. Be sure to use a protective mitt when handling the
flask containing the agarose solution.
8. Once the agarose is completely dissolved, remove the flask from the microwave using a protective mitt and let
cool (~10 minutes) until you can hold your hand to the flask for at least 10-15 seconds.
9. Once cooled, carefully pour the agarose into the gel tray and insert the comb in the middle of the gel before
the gel starts to polymerize.
10. Allow the gel to set for approximately 20 minutes or until it has hardened.
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Gel electrophoresis; Load, run, and examine the gel:
11. Once the gel has completely hardened, place it in the electrophoresis box and add enough 1X TAE buffer to
the electrophoresis chamber to submerge the gel.
12. Carefully wiggle the comb to loosen it and slowly remove the comb from the gel. Use caution when removing
the comb as it is easy to damage the gel.
13. Using a p20, load 10 µL of each of your samples onto the gel wells in the following order. Be sure to use a
fresh tip for each tube!
Lane 1
Bromophenol blue (BB)
Lane 2
Methyl orange (MO)
Lane 3
Janus green (JG)
Lane 4
Xylene cyanol (XC)
Lane 5
Safranin O (SO)
Lane 6
Fuschsin basic (FB)
Lane 7
Unknown A
Lane 8
Unknown B
14. Place the cover on the electrophoresis box.
15. Connect the electrodes from the gel box to the power supply – the red lead should be connected to the
positive electrode and the black lead to the negative electrode.
16. Turn on the power supply and set the voltage to ~125 volts. Check to confirm samples are moving away from
the wells. DO NOT OPEN THE GEL BOX LID WHILE CURRENT IS RUNNING THROUGH THE GEL BOX!
17. Run the gels at constant voltage for five minutes and note what you see in the lanes for unknown A and B.
I see…_________________________________________________________________________.
18. Continue to run your gel until the dye front is about 2/3rds of the way down from the top of the gel – about 20
minutes. Turn off the power supply, disconnect the electrodes, and carefully remove the gel from the box.
19. Use a ruler to draw your results on gel the template below as accurately as possible; if possible take a
photograph of your gel and attach it to your to this sheet.
Sample:
BB
MO
JG
XC
SO
FB
A
B
20. Compare the bands seen in the lanes for Unknown A and Unknown B with the bands seen in lanes 1-6.
Can you determine the mixture of dyes in each unknown? Unknown A =___________________,
Unknown B =___________________.
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Molecular Rainbow: Electrophoresis to Identify the
Molecules in a Mixture
Pre-Lab Questions
Directions: After reading through the introduction and protocol for the Molecular Rainbow lab, answer the
questions below.
1. Understanding How does the agarose gel electrophoresis work to separate molecules for one another? What
is the force that cause the molecules to move?
2. Understanding What is the role of the TAE in this experiment?
3. Understanding What would happen to the dye molecules in the gel if, during the experiment, the cord to the
positive pole was not attached?
4. Applying You observe the following while performing gel electrophoresis on two molecules at the same time
and under the same conditions. Although the molecules are the same size you observe that molecule 1 moves 2
cm towards the positive pole while molecule 2 barely moves from the well. Give at least one explanation to
explain why these two molecules of the same size behave differently in your experiment.
5. Applying You observe the following while performing two gel electrophoresis experiments of the same
molecule, molecule A. You run the gels for the same amount of time but you find that in one gel, molecule A
moves 3 cm towards the positive pole while in a different gel molecule A moves 4 cm towards the positive pole.
Give at least two explanations that could explain why the molecule A migrated a different distance in the same
amount of time in the two experiments.
6. Understanding Explain why it is hard to guess the mixture in unknown A or B by comparing the color of each
to the known dyes.
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Molecular Rainbow: Electrophoresis to Identify the
Molecules in a Mixture
Post-Lab Questions and Analysis
Directions: After completing the Molecular Rainbow lab, answer the questions below.
1. Analyzing You probably could not determine the mixtures in unknown A and B at five minutes, but you likely
could determine the mixtures in unknown A and B at the end of your experiment. Explain why running the gel for
more time gave you more information.
2. Analyzing
Unknown A was a mixture of which dyes?
Unknown B was a mixture of which dyes?
3. Applying You have a tube of purple dye that is either Cresol blue or Thymol blue, but you do not know which.
Assume you have all of the materials for the lab you just completed. Remember, you only one unknown tube with
either Cresol blue or Thymol blue, not both. You do not have a stock so you cannot compare the two. Size and
charge of the dyes you used today are given in the shaded area of the table below. The size and charge of
Cresol blue and Thymol blue are given in the unshaded area of the table below.
Biological dye
Formula Weight
Bromophenol blue
692
Methyl orange
328
Janus green
511
Xylene cyanol
539
Safranin O
351
Cresol blue
Thymol blue



490
646
Charge
+
+
Color in TAE
Blue
Yellow
Blue
Blue
Red
-
Purple
Purple
Design a gel electrophoresis experiment that will allow you to determine if the unknown purple dye is
Cresol blue or Thymol blue. Label the wells of the gel below with the samples you plan to run in each
well. Strive to use the fewest possible lanes that will allow you to identify the unknown purple dye.
Sketch what you would see on your gel after the electrophoresis experiment is complete.
Describe the data you would obtain from the gel that would allow you to identify the unknown purple dye.
_
+
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