Molecular Rainbow: Dye Electrophoresis to Identify the
Molecules in a Mixture
Teacher Materials
Leaning Goals, Objectives, and Skills ................................................................ 2
Standards Alignments .......................................................................................... 3
Laboratory Set-Up Manual ................................................................................... 4
Instructor Laboratory Guide ................................................................................ 7
Answers to Student Questions............................................................................ 8
Molecular Rainbow: Dye Electrophoresis to Identify the
Molecules in a Mixture
Students Learning Goals:
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Students will understand that molecules vary in size, shape and charge.
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Students will understand what gel electrophoresis is how it is used to separate molecules bases upon size
and charge.
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Students will understand how to use concentrated stock solutions to make dilute working solutions.
Student Learning Objectives:
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Students will analyze the results of the molecule separation by gel electrophoresis.
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Students will assign relative the size of dye molecules as a function of movement through the agarose gel.
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Students will determine the components of an unknown dye mixture.
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Students will predict how additional molecules would have migrated in gel electrophoresis based upon size
and charge.
Scientific Inquiry Skills:
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Students will pose questions and form hypotheses.
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Students will design and conduct scientific investigations.
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Students will use experimental data to make conclusions about the initial question and to support or refute
the stated hypothesis.
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Students will follow laboratory safety rules and regulations.
Laboratory Technical Skills:
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Students will demonstrate proper use of micropipettors.
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Students will consider safety considerations when working with an electric current.

Students will make dilute solutions from concentrated stock solutions

Students will demonstrate proper use of gel electrophoretic equipment.

Students will prepare and pour agarose gels.
2
Molecular Rainbow: Dye Electrophoresis to Identify the
Molecules in a Mixture
Standards Alignments
MA Science and Technology/Engineering Curriculum Framework (2006)
Under Development
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Molecular Rainbow: Dye Electrophoresis to Identify the
Molecules in a Mixture
Laboratory Set-Up Manual
Set-up assumes 30 students, working in groups of two, for a total of 15 stations.
Supply List:
For lab preparation:
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15 Electrophoresis gel boxes, trays, combs, and electrodes
Power supplies (number depends on how many gels each can support)
15 X p20 Micropipettors and tips
150 X 1.5 mL Microcentrifuge tubes (this includes some extras)
18 X 50 mL Conical Tubes
15 X 50 mL Tube racks
15 X 50 mL Graduated cylinders
15 X 500 mL Graduated cylinders
15 X 250 mL Erlenmeyer flasks
15 X 500 mL Erlenmeyer flasks
15 Rulers
Agarose – 10 g (Fisher Scientific catalog #: BP1356-100)
10X tris-acetate buffer – 900 mL (Fisher Scientific catalog # 50-843-235)
Dyes – see Table 1 for amounts
o Bromophenol blue (Fisher Scientific)
o Methyl orange (Fisher Scientific)
o Janus green (Fisher Scientific)
o Xylene cyanol (Fisher Scientific)
o Safranin O (Fisher Scientific)
o Fuschsin basic (Fisher Scientific)
2 X 4 L carboys
microwave and potholders, gloves, etc.
For each group:
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15 Electrophoresis gel boxes, trays, combs, and electrodes and 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
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Set-up Calendar:
2 weeks before lab:
 Check supplies and order any needed materials
1-3 days before lab:
 Fill 2 X 4 L carboys with dH2O
 Aliquot 50 mL of 10X TAE into 18 X 50 mL conical tubes – store at RT. Confirm amount is sufficient to make
enough running buffer for the gel boxes students are using.
 Prepare the Standard Dye samples according to Table 1
Table 1. Standard Dye Samples
Standard
Solution
Bromophenol blue
Methyl orange
Janus green
Xylene cyanol
Safranin O
Fuschsin basic
Powdered Dye
BB
MO
JG
XC
SO
FB
4 mg
10 mg
100 mg
4 mg
4 mg
4 mg
60% Glycerol
Solution*
10 mL
10 mL
10 mL
10 mL
10 mL
10 mL
Final
Concentration
0.04%
0.10%
1.00%
0.04%
0.04%
0.04%
* The 60% Glycerol Solution can be substituted with 4 g of sucrose and 10 mL of dH 2O
** If starting with a liquid dye, dilute with 60% glycerol solution to appropriate concentration
 Prepare the Unknown dye samples according to Table 2
Table 2. Unknown Dye Samples
Unknown A
1 mL Bromophenol blue
1 mL Methyl Orange
Unknown B
1 mL Xylene cyanol
1 mL Safranin O
*If you choose to alter the unknown solutions by substituting other mixtures of dyes, be sure to not mix two dyes
that will migrate the same distance.
 Aliquot 12 L of each standard dye sample into 18 X 1.5 mL microcentrifuge tubes, label as follows and store
at room temperature:
1 day before lab:
 Set up 15 student stations. Each station should have the following lab materials:
 Electrophoresis gel box, tray,
 500 mL Erlenmeyer flask
comb, and electrodes
 Permanent marker
 Power supply
 Ruler
 p20 Micropipettor and tips
 10X TAE buffer
 50 mL Tube rack
 6 Standard Dye samples
 50 mL Graduated cylinder
 2 Unknown samples
 500 mL Graduated cylinder
 250 mL Erlenmeyer flask
 Set up weighing stations (at least four)
Electronic balance
Weighing paper or boats
Tongue depressors or spatulas
Agarose ~ 25 g in a 50 mL conical tube
 Check microwave and potholders
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Set-up Calendar, continued :
2 weeks before lab:
 Check supplies and order any needed materials.
 If making any substitutions to the supply list, edit the student protocol accordingly.1 day before lab
 Set up student lab stations with all durable materials.
 Prepare TAE buffer
Morning of lab:
 Prepare 1.8% agarose gel mix
 Aliquot out the plasmid DNA, ladder DNA, loading dye, dH2O, buffer. Keep all tube on ice or in freezer.
 Prepare 3 ice buckets (1 for every 5 groups)
1. Aliquot 10 L of each enzyme into its own microcentrifuge tube. Keep all tubes on ice
2. Pour agarose gels. 1 gel/group
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Molecular Rainbow: Dye Electrophoresis to Identify the
Molecules in a Mixture
Instructor Laboratory Guide
Laboratory Procedure Tips:
1. Many different biological dyes and stains may be used in this lab. Many dyes and stains are also acid-base
indicators; as such their color will change depending on the pH of the solution. Please refer to a reference scientific
manual or catalog for more information about any indicator that you plan to use. It is also a good idea to test each
one first to see how well they work before using with students. Table 3 lists some common biological dyes and stain,
their formula weights and charges, the recommended concentrations, and the color of each dye in tris-acetate buffer.
Table 3 source: Flinn Scientific “Dyeing for Electrophoresis” Lab:http://www.flinnsci.com/Documents/demoPDFs/Biology/BF10901.pdf
2. Abbreviations:
Bromophenol blue
Methyl orange
Janus green
Xylene cyanol
Safranin O
Fuschsin basic
Unknown A
Unknown B
BB
MO
JG
XC
SO
FB
A
B
3. Before starting the lab…
 Ask students to check their materials list to make sure they have everything.
 Demonstrate how to pipet very small volumes of liquid.
 Remind students to use a fresh pipet tip between each addition.
4. If students alter the loading order it does not matter! Just make sure they record the order they used.
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Molecular Rainbow: Dye Electrophoresis to Identify the
Molecules in a Mixture
Answers to Student Questions
Protocol-Embedded:
p. 2: Sample answers:
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The mouse would reach the destination first. The mouse is small and the one foot spacing between the
trees does not pose an obstacle to the mouse, but it does to you.
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Molecules with a negative surface charge will move towards to positive pole, molecules with a postive
surface charge will move towards to negative pole.
p. 3:
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If you want to make 20 ml of 1X TAE buffer, you would mix 2 ml of 10X TAE with 18 ml water.
20ml (1x) = ____ ml (10x); 20ml (1x)/(10x) = 2 ml. 20ml (final volume) – 2 ml TAE = 18 ml water.
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If you want to make 120 ml of 1X TAE buffer, you would mix 2.4 ml of 50X TAE with 117.6 ml water.
120ml (1x) = ____ ml (50x); 120ml (1x)/(50x) = 2.4 ml. 120ml (final volume) – 2.4 ml TAE = 117.6 ml water.
p. 4: Expected answer:
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Unknown A is a mixture of Bromophenol blue and Methyl Orange. Unknown B is a mixture of Xylene cyanol
and Safranin O.
Pre-Lab:
1. The agarose gel forms a matrix or mesh that acts as an obstacle course for molecules. Different sized
molecules move through the gel at different rates, depending upon their size. Small molecules can move
more quickly through the gel than can large molecules. An electric field applied which causes the molecules
move through the gel. Positive molecules move towards the negative pole and negative molecules move
toward the positive pole.
2. TAE is a buffered salt solution that controls the pH and provides the ions needed to carry the electric current.
3. If the cord to the positive pole was not attached, then there would be no electric field or current and the dye
molecules would barely move through the gel. The molecules would move a little bit due to diffusion.
4. If the two different molecules of the same size had different surface charge, then they would behave
differently in your experiment. In this case, molecule 1 moves towards the positive pole, so it would have a
negative surface charge. Molecule 2 barely moves from the wells, so it would have little or no surface
charge.
5. Option 1) the two gels have different percentages of agarose, where one has a significantly higher
percentage than the other. If one gel with a higher percentage of agarose, it will have a matrix that is
denser, and molecules will travel more slowly in the same amount of time. Option 2) the voltage applied to
the two gels was different. The higher the applied voltage, the faster the molecules migrate.
6. It is difficult to use color alone because each unknown is a mixture and as such, it would be hard to tell if a
purple color is a combination of blue and red dyes or whether it is due to lots of purple dye.
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Post-Lab and Analysis:
1. The idea is analogous to that of footrace, like the Boston marathon. At the beginning and for the first few
miles, everyone is clustered together, but by mid race and beyond, there is great distances between the fast
runners and the slow runners.
2.
Unknown A is a mixture of Bromophenol blue and Methyl Orange. Unknown B is a mixture of Xylene cyanol
and Safranin O.
Xylene cyanol
Unknown purple dye
3. Sample answer: You will need to compare the known dyes to the unknown dyes, much like you did in
today’s lab. However, you can only compare relative size, not color. Remember, you only have Cresol blue
or Thymol blue, not both, so you cannot compare the two. Bromophenol blue is larger than either Cresol
blue or Thymol blue so comparing your unknown to Bromophenol blue will tell you that the unknown is
smaller but not how much smaller. Likewise, Methyl orange is smaller than either Cresol blue or Thymol blue
so comparing your unknown to Methyl orange will tell you that the unknown is larger but not how much
larger. The size of Xylene cyanol is between that of Cresol blue or Thymol blue, so Xylene cyanol is the
ideal comparison. If the unknown purple dye migrates further than Xylene cyanol, you would identify it as
Cresol blue. If the unknown purple dye migrates a shorter distance than Xylene cyanol, you would identify it
as Thymol blue.
646
or
539
516
+
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