Electrophoresis

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Electrophoresis Powered By Docstoc
					Using Electrophoresis and other Separation Techniques in
                  High School Science

                            Glenn Voshell
                         Colton High School
                             Colton, WA
                                  &
                            Martin Nuxoll
                      Lewis-Clark State College
                            Lewiston, ID


                Washington State University Mentors
                            Dr. Neil Ivory
                        Chemical Engineering
                                  &
                             Jeff Burke
                          Graduate Student


        A project supported by the National Science Foundation
                       Grant No. EEC-0338868




                                  1
                                       Project Summary
This module was designed to introduce separation techniques while incorporating biotechnology
into the classroom. Guided inquiry techniques and solid scientific practices are employed
throughout the module. The use of proteins (or simulation using dyes) and DNA allows this six
to ten day module to fit nicely into a Genetics Unit in an introductory Biology course. This
module introduces the use of basic techniques to teach concepts including solubility,
electrophoretic mobility and molecular weight. These basic techniques and concepts are often
used by Chemical Engineers to separate various biomolecules for improving scientific
knowledge and solving many of the biomedical challenges society encounters.

Various components of the module could be directly incorporated at different levels and different
subject areas in the junior high or high school curriculum. All module activities are accompanied
by a corresponding appendix, providing insight to the teacher about where that activity might be
best used and recommendations that should minimize the preparation time necessary for the
activity. This document was prepared using Microsoft Word. Please feel free to download and
modify it to meet the needs of your individual classroom.

                                          Introduction
Science and technology now advance at an extremely fast rate. Science teachers have the
difficult job of trying to teach everything that has been historically taught while still finding time
to teach all the new things. The only realistic way to accomplish this is to find ways to
incorporate biotechnology into the basic curriculum so both needs are met. Education reform
efforts around the nation have added to the pressure on teachers to find or develop multi-faceted
curricula which blends the old with the new. The authors attempted to design this module to
combine basic concepts by using current biotechnology appropriate for the secondary classroom.

The underlying concepts studied by scientists and engineers are similar but what they do with
that information varies. Scientists tend to study things to understand them (hypothetico-
deductive), while engineers want to understand things so they can apply that knowledge to
accomplish a particular task (inductive).

The authors worked cooperatively with engineers to learn the underlying scientific principles and
understand how engineers apply those principles to solve real life problems. The authors hope
their experience in this project, and the development of the accompanying module will foster an
interest in science, engineering, and related fields in junior high and high school students.

                                        Goals of Project

General Goals

The primary goal of this module was to blend use of current technology with instruction in the
basic concepts in separation, DNA, and forensic techniques. A second goal was to provide
students with experiences that relate to basic principles of engineering.




                                                  2
Specific Student Goals

At the conclusion of this module, students should be able to:

1. Accurately use a micropipet.
2. Set up, run, and interpret the results of an agarose gel experiment.
3. Analyze components of dyes (proteins), in terms of solubility and electrophoretic mobility
   using appropriate techniques.
4. Analyze components of DNA in terms of molecular weight.
5. Determine electrophoretic velocities and apply to the identification of an unknown.




                                                3
                                    Micropipet Practice Activity

(See Appendix A for teacher instructions and suggestions)
This activity was adapted from "High School Science and Biotechnology:
Conceptual Development of Electrophoresis" by Carole Bennett & Brian Hardcastle (1995)
[See URL for activity: http://www.che.wsu.edu/home/modules/.]

Purpose

To teach students the proper operation of a micropipet.

Materials

Kit with foam block holding plastic microcentrifuge tubes of red, green, blue and yellow food
dyes
4 microcentrifuge tubes (1.5mL)
pipet tips
filter paper
micropipet

Procedures

Use of micropipet volume setting

1. Look at micropipet to determine the volume range: 0.5 µL - 10 µL or 2 µL- 20 µL.
2. Find out from your instructor if this pipet has a volume lock setting. If so, release the lock as
   shown.
3. Turn the control knob to select the needed volume. DOUBLE CHECK IT.
4. Attach a pipet tip. Handle the pipet with thumb on the release button and in a vertical
   position. TO AVOID CONTAMINATION OR DAMAGING THE PIPET, NEVER
   PIPET LIQUID WITHOUT ATTACHING A TIP TO THE PIPET. NEVER LAY
   PIPET DOWN WITH LIQUID IN THE TIP.

Filling the pipet

5.   Press the control button down to the first stop.
6.   Immerse the pipette tip 2-mm into the liquid.
7.   Allow the control button to glide back slowly.
8.   Slide the tip out along the inside of the container.
9.   Wipe off any external droplets on the tip with lint-free tissue.

Dispensing

10. Immerse tip into the liquid.
11. Slowly press the control button completely to activate the blow-out feature.
12. Eject the tip.


                                                   4
13. Practice using the pipet with samples of water to become comfortable with the feel of the
    control button stops.




Formation of four new colors

14. Place a 3 µL sample of the basic dye colors (red, green, blue & yellow) on filter paper and
    label spots with pencil.
15. Use the chart below to mix the required µL of basic colors in correct clean, empty,
    microcentrifuge tubes.

                                       Red Green Blue Yellow
                          Teal             5 µL 15 µL
                          Rose       15µ L       5 µL
                          Orange     6µ L             14 µL
                          Chartreuse       2 µL       18µ L

16. Mix all dyes into a single droplet in the bottom of each microcentrifuge tube. (Use vortex
    mixer or a clean, plastic toothpick)
17. Place a 3 µL sample of the mixed colors on the same piece of filter paper that you placed the
    basic colors.

Cleaning up

18. Wash the microfuge tube with the mixed colors (teal, rose, orange & chartreuse) with
    distilled water.
19. Replace the clean microcentrifuge tubes in kit for next group.
20. Clean up area.
21. Replace micropipet as indicated by your instructor.
22. Attach the filter paper to a sheet of paper with your name and date for grading.



Discussion Questions

1. Compare the sizes of dye spots on your filter paper. The sizes of the spots should be identical
   if your technique is correct.
2. Compare the colors produced for teal, rose, orange and chartreuse with a standard. This also
   indicates your ability to follow directions and handle the micropipet correctly.
3. How could you use a metric scale to check the calibration of the micropipet.




                                                5
                              Extraction of Ink from Paper
(See Appendix B for teacher instructions and suggestions)

Purpose

To extract ink from a note to be further analyzed via various separation techniques.

Materials

Small test tubes or microcentrifuge tubes (1.5 mL is ideal)
Handwritten note (teacher provided)
Micropipet
Distilled water
Scissors

Procedures

1. Cut letters out of note, attempting to retrieve only inked portions of the paper.
2. Place at least 5 inked letters into a small test tube.
3. Micropipet 150 L of distilled water into the small test tube.
4. Verify that all letters are submersed (under) in water.
5. Allow to set for 5 minutes.
6. Record observations.
7. Remove the ink/water sample by pipetting out as much of the liquid as possible from the test
   tube and placing it into another tube or microcentrifuge tube.
8. Clean the small test tube

Discussion Questions

1. What advantage was gained by carefully trimming the letters?
2. Why do you think there was a small amount of ink remaining (a faint outline) on the paper
   from which the ink was extracted?
3. Suppose your ink/water sample was too dilute. Propose two ways to concentrate it.




                                                6
                                Thin Film Chromatography
(See Appendix C for teacher instructions and suggestions)

Purpose

To demonstrate how components of an ink sample will separate according to their solubilities
when moving up a vertically suspended piece of filter paper.

Materials

4" diameter filter paper
250mL beaker
deionized water or buffer
pencil
scotch tape
ruler
ink sample or ink pen

Procedures

1. Cut filter paper into 1-2cm wide strips, cutting one end to a point.
2. Apply a small spot (3-5mm diameter) of ink or dye 1cm up from the pointed end.
3. Pour solvent (water or buffer) into beaker until approx. 0.5 cm deep.
4. Suspend filter paper strip from pencil so that no part of the strip touches the glass beaker, and
    only the point is in the solvent. (The ink spot must not be directly in the solvent.)
5. Bend filter paper strip over pencil and use tape to secure in position.
6. Wait until the colored bands of ink have separated and stopped moving (approx. 10 min.)
7. Remove strip and allow it to air dry.
8. Measure and record the color and distance that each band moved from the starting point
    (Measure from estimated center of colored band).
9. Save and attach colored filter paper strip to lab report.
10. Record any other observations.
11. Clean beaker and lab area.

Discussion Questions

1. What mechanism is responsible for the solvent traveling up the strip? What other phenomena
   are explained by the same principle?
2. Why did the colored bands separate at different locations on the paper?
3. What would you expect to happen to the number of bands if you were to use a red pen?




                                                 7
                               Agarose Gel Electrophoresis
(See Appendix D for teacher instructions and suggestions)

Purpose

To demonstrate how components of a dye sample will separate according to their charges.

Materials

power supply
gel box
agarose
deionized water
(1X) TAE buffer
ruler
dye samples
micropipet
wick

Procedures

1. If your instructor has prepared your agarose gel for you, skip to step 2. If preparing your own
    agarose gel, weigh out 0.25g of agarose. Add this to 25 mL of 1X TAE buffer in a 100mL
    beaker. Heat on a hot plate (stirring constantly) or in microwave (initial time of 20 seconds,
    then stir, subsequent times no longer than 10 seconds) until all bubbles are gone. DO NOT
    ALLOW AGAROSE TO BOIL OVER.
2. Set up the gel box for pouring by placing the black metal dams beside the gel plate in the
    appropriate slots.
3. Slowly pour agarose gel onto gel plate, making sure that the entire surface of the gel plate is
    covered. Remove any bubbles using a stir rod.
4. Place the eight tooth white comb into the center slot.
5. Allow gel to cool until it solidifies (~5minutes). While waiting, hook up wires to the power
    supply. Make sure you have a micropipet, tips, and samples ready.
6. Carefully remove the comb from the gel.
7. Follow the instructions for an immersion or a wick gel according to your instructor's
    directions.
Immersion gel: Add buffer into buffer reservoirs in gel box until gel is submersed. (DO NOT
    POUR BUFFER DIRECTLY ONTO GEL SURFACE BECAUSE IT COULD
    DAMAGE THE GEL).
Wick gel: Cut a piece of wicking material (if not cut in advance) into two 6cm x 6cm squares.
    Place the wick on each side of the gel box assuring that the wick covers the entire width of
    the gel but only covers ~0.5cm of the surface of the gel. The other end of the wick should be
    placed such that it rests on the bottom of the buffer reservoir. Repeat with other side of gel
    box. Pour buffer into each buffer reservoir until they are full (do not allow the buffer from




                                                8
   one reservoir to run underneath the gel plate and contact the other buffer). The wick material
   should absorb the buffer, so you may have to add a little more. (See figure D.1)




FIGURE D.1

8. Place a tip on the micropipet. Set for the appropriate volume. Push the control button down to
   the first stop. Place the pipette tip at least 2-mm into the sample. Let the control button move
   back slowly.
9. Load 10µL of sample into a well on the edge of the gel. Immerse tip into the well in the gel
   (See figure D.2). Slowly press the control button to the second stop (this activates the blow-
   out feature assuring all the sample has been dispensed). Since we are using the same sample,
   the tip does not have to be removed after each loading. Each student should load a sample
   into a well (leave an empty well between each student sample).




FIGURE D.2

10. Close the gel box lid and insert the wires into their respective plugs.
11. Turn power supply on. Set voltage at 100 V. TO AVOID ELECTRIC SHOCK, DO NOT
    TOUCH GEL BOX OR WIRES WHILE THE POWER IS ON. Record start time.


                                                9
12. Observe and record any activity within the gel.
13. When fastest band is within ~1cm of the gel edge, turn power supply off. Record stop time.
14. Measure (in cm) the distance each band has traveled and record their colors. Make a
    drawing of your gel, with distance and colors accurately reflected.

Discussion Questions

1. Why did the colored bands separate at different locations in the gel?
2. What would you expect to happen to the rate at which the bands traveled if you increased the
    voltage? What would you expect to happen to the rate at which the bands traveled if we
    increased the concentration of the buffer?
3. Did you notice any difference in any of the samples (since everything should have been the
    same)? If so, what may have accounted for this difference?
4. If you used the immersion technique:
    Why was it necessary for you or your instructor to add glycerol to the dye sample?




                                              10
                    Chromatography/Electrophoresis Comparison
(See Appendix E for teacher instructions and suggestions)

Purpose

To compare how various components of a dye sample will separate differently based upon the
separation technique

Materials

4" diameter filter paper
250mL beaker
pencil
scotch tape
ruler
power supply
gel box
agarose
(1X) TAE buffer
ruler
dye samples
micropipet
wick

Procedures

1. If your instructor has prepared your agarose gel for you, skip to step 2. If preparing your own
    agarose gel, weigh out 0.25g of agarose. Add this to 25 mL of 1X TAE buffer in a 100mL
    beaker. Heat on a hot plate (stirring constantly) or in microwave (initial time of 20 seconds,
    then stir, subsequent times no longer than 10 seconds) until all bubbles are gone. DO NOT
    ALLOW AGAROSE TO BOIL OVER.
2. Set up the gel box for pouring by placing the black metal dams beside the gel plate in the
    appropriate slots.
3. Slowly pour agarose gel onto gel plate, making sure that the entire surface of the gel plate is
    covered. Remove any bubbles using a stir rod.
4. Place the eight tooth white comb into the center slot.
5. Allow gel to cool until it solidifies (~5mins). While waiting proceed with step 6.
6. Cut filter paper into 1-2cm wide strips, cutting one end to a point
7. Apply a small spot (3-5mm diameter) of ink or dye 1cm up from the pointed end. If spot does
    not appear dark, let it dry and add another spot until it is darker, repeat as necessary.
8. Suspend filter paper strip from pencil so that no part of the strip touches the glass beaker, and
    the point barely touches the bottom of the beaker.
9. Bend filter paper strip over pencil and use tape to secure in position.
10. Remove filter paper strip and pencil, pour buffer about 0.5cm deep in beaker.
11. Go back to gel, make sure it is set up. Carefully remove comb from gel.


                                                11
12. Cut a piece of wicking material (if not cut in advance) into two 6cm x 6cm squares.
    Place the wick on each side of the gel box assuring that the wick covers the entire width of
    the gel but only covers ~0.5cm of the surface of the gel. The other end of the wick should be
    placed such that it rests on the bottom of the buffer reservoir. Repeat with other side of gel
    box. Pour buffer into each buffer reservoir until they are full (do not allow the buffer from
    one reservoir to run underneath the gel plate and contact the other buffer). The wick material
    should absorb the buffer, so you may have to add a little more. (See figure E.1)




   FIGURE E.1

13. Load 10µL of sample into a well on the gel. Load additional wells (let other partners
    participate here) if you have enough dye.
14. Close the gel box lid and insert the wires into their respective plugs.

STEPS 15 & 16 NEED TO BE DONE SIMULTANEOUSLY.

15. Turn power supply on. Record start time. Set voltage at 100 V. TO AVOID ELECTRIC
    SHOCK, DO NOT TOUCH GEL BOX OR WIRES WHILE THE POWER IS ON.
16. Insert filter paper into solvent (buffer). Be sure solvent is below dye spot.
17. Observe and record any activity within the gel and on the filter paper.
18. When fastest band is within ~1cm of the gel edge, turn power supply off. Remove filter
    paper and record stop times.
19. Allow strip to air dry.
20. Measure (in cm) the distance each band has traveled and record their colors. Make a
    drawing of your gel and filter paper, with distance and colors accurately reflected (measure
    to center of colored band).
21. Save and attach colored filter paper strip to lab report.
22. Buffer can be recycled. Clean beaker and lab area.




                                                12
Discussion Questions

1. In what order did the colored bands move (slowest to fastest) in the gel and on the filter
    paper?
2. How would you explain the difference you observed in each procedure (see question #1)?
    What mechanisms accounted for the separation in each case? HINT: Think about what
    variables were different.
3. What would you expect to happen to the rate at which the bands traveled if you increased the
    voltage? What would you expect to happen to the rate at which the bands traveled if we
    increased the concentration of the buffer?
4. Did you notice any difference in any of the samples (since everything should have been the
    same)? If so, what may have accounted for this difference?




                                              13
                         Determining Electrophoretic Velocity
(See Appendix F for teacher instructions and suggestions)

Purpose

To determine the electrophoretic velocity of various dyes.

Materials

power supply
gel box
agarose
deionized water
(1X) TAE buffer
ruler
dye samples
micropipet
wick

Procedures

1. If your instructor has prepared your agarose gel for you, skip to step 2. If preparing your own
    agarose gel, weigh out 0.25g of agarose. Add this to 25 mL of 1X TAE buffer in a 100mL
    beaker. Heat on a hot plate (stirring constantly) or in microwave (initial time of 20 seconds,
    then stir, subsequent times no longer than 10 seconds) until all bubbles are gone. DO NOT
    ALLOW AGAROSE TO BOIL OVER.
2. Set up the gel box for pouring by placing the black metal dams beside the gel plate in the
    appropriate slots.
3. Slowly pour agarose gel onto gel plate, making sure that the entire surface of the gel plate is
    covered. Remove any bubbles using a stir rod.
4. Place the eight tooth white comb into the center slot.
5. Allow gel to cool until it solidifies (~5mins). While waiting, hook up wires to the power
    supply. Make sure you have a micropipet, tips, and samples ready.
6. Carefully remove the comb.
7. Follow the instructions for an immersion or a wick gel according to your instructors
    directions.
Immersion gel: Add buffer into buffer reservoirs in gel box until gel is submersed. DO NOT
    POUR BUFFER DIRECTLY ONTO GEL SURFACE TO AVOID DAMAGING THE
    GEL.
Wick gel: Cut a piece of wicking material (if not cut in advance) into two 6cm x 6cm squares.
    Place the wick on each side of the gel box assuring that the wick covers the entire width of
    the gel but only covers ~0.5cm of the surface of the gel. The other end of the wick should be
    placed such that it rests on the bottom of the buffer reservoir. Repeat with other side of gel
    box. Pour buffer into each buffer reservoir until they are full (do not allow the buffer from




                                                14
   one reservoir to run underneath the gel plate and contact the other buffer). The wick material
   should absorb the buffer, so you may have to add a little more.

8. Load 10µL of sample into a well on the edge of the gel. Immerse tip into the well in the gel.
    Slowly press the control button to the second stop (this activates the blow-out feature
    assuring all the sample has been dispensed). Repeat loading of each of the dyes specified by
    instructor (If numerically possible, leave an empty well between dyes).
9. Close the gel box lid and insert the wires into their respective plugs.
10. Turn power supply on. Set voltage at 100 V. TO AVOID ELECTRIC SHOCK, DO NOT
    TOUCH GEL BOX OR WIRES WHILE THE POWER IS ON. Record start time.
11. Observe and record any activity within the gel.
12. When fastest band is within ~1cm of the gel edge, turn power supply off. Record stop time.
13. Measure (in cm) the distance each band has traveled and record their colors. Make a
    drawing of your gel, with distance and colors accurately reflected.

Discussion Questions

1. Determine the velocity (in cm/min) for each dye sample being sure to specify the direction of
   travel for each.
2. What would you expect to happen to the electrophoretic velocity of the bands if you increased
   or decreased the voltage?
3. What would you expect to happen to the electrophoretic velocity of the bands if you increased
   the concentration of the buffer?
4. How would a change in the concentration of the agarose affect the electrophoretic velocity?
5. What causes dyes or proteins to move in opposite directions?
6. You may have noticed that the bands of dye spread out some before and/or after you ran the
   gel. Explain why you think the bands spread.




                                               15
Identifying Unknowns Using Electrophoretic Velocity

(See Appendix G for teacher instructions and suggestions)

Purpose

To identify unknown dyes or proteins using electrophoretic velocity.

Materials

power supply
gel box
agarose
deionized water
(1X) TAE buffer
ruler
unknown samples or mix
micropipet
wick

Procedures

1. If your instructor has prepared your agarose gel for you, skip to step 2. If preparing your own
    agarose gel, weigh out 0.25g of agarose. Add this to 25 mL of 1X TAE buffer in a 100mL
    beaker. Heat on a hot plate (stirring constantly) or in microwave (initial time of 20 seconds,
    then stir, subsequent times no longer than 10 seconds) until all bubbles are gone. DO NOT
    ALLOW AGAROSE TO BOIL OVER.
2. Set up the gel box for pouring by placing the black metal dams beside the gel plate in the
    appropriate slots.
3. Slowly pour agarose gel onto gel plate, making sure that the entire surface of the gel plate is
    covered. Remove any bubbles using a stir rod.
4. Place the eight tooth white comb into the center slot.
5. Allow gel to cool until it solidifies (~5minutes). While waiting, hook up wires to the power
    supply. Make sure you have a micropipet, tips, and samples ready.
6. Carefully remove the comb.
7. Follow the instructions for an immersion or a wick gel according to your instructors
    directions.
Immersion gel: Add buffer into buffer reservoirs in gel box until gel is submersed. DO NOT
    POUR BUFFER DIRECTLY ONTO GEL SURFACE TO AVOID DAMAGING THE
    GEL.
Wick gel: Cut a piece of wicking material (if not cut in advance) into two 6cm x 6cm squares.
    Place the wick on each side of the gel box assuring that the wick covers the entire width of
    the gel but only covers ~0.5cm of the surface of the gel. The other end of the wick should be
    placed such that it rests on the bottom of the buffer reservoir. Repeat with other side of gel
    box. Pour buffer into each buffer reservoir until they are full (do not allow the buffer from




                                                16
   one reservoir to run underneath the gel plate and contact the other buffer). The wick material
   should absorb the buffer, so you may have to add a little more.

8. Load 10µL of unknown sample into a well on the edge of the gel. Immerse tip into the well in
    the gel. Slowly press the control button to the second stop (this activates the blow-out feature
    assuring all the sample has been dispensed). Repeat loading of each of the dyes specified by
    instructor (If numerically possible, leave an empty well between dyes).
9. Close the gel box lid and insert the wires into their respective plugs.
10. Turn power supply on. Set voltage at 100 V. (TO AVOID ELECTRIC SHOCK, DO NOT
    TOUCH GEL BOX OR WIRES WHILE THE POWER IS ON) Record start time.
11. Observe and record any activity within the gel.
12. When fastest band is within ~1cm of the gel edge, turn power supply off. Record stop time.
13. Measure (in cm) the distance each band has traveled and record their colors. Make a
    drawing of your gel, with distance and colors accurately reflected.

Discussion Questions

1. Determine the velocity (in cm/min) for each color band in the dye sample being sure to
   specify the direction of travel for each.
2. What dyes or proteins were present based upon your comparison of your electrophoretic
   velocities with the velocities determined in the previous lab activity?
3. Why is it of critical importance to maintain the exact conditions between this unknown lab
   and the previous lab where you determined the electrophoretic velocities of the known dyes
   or proteins?
4. Why is keeping the length of time the gel is run not important in this case?
5. Discuss three conditions that would alter electrophoretic velocity if they were changed.




                                                17
                         Agarose Gel Electrophoresis of Precut DNA

(See Appendix H for teacher instructions and suggestions)

Purpose

To demonstrate the role of molecular weight in electrophoretic separation.

Materials

power supply
gel box
agarose
Carolina BLU Gel & Buffer Stain
(1X) TAE buffer
Carolina BLU Final DNA Stain
deionized water
ruler
Precut DNA
micropipet
wick

Procedures

1. If your instructor has prepared your agarose gel for you, skip to step 2. If preparing your own
    agarose gel, weigh out 0.25g of agarose. Add this to 25 mL of 1X TAE buffer in a 100mL
    beaker. Heat on a hot plate (stirring constantly) or in microwave (initial time of 20 seconds,
    then stir, subsequent times no longer than 10 seconds) until all bubbles are gone. DO NOT
    ALLOW AGAROSE TO BOIL OVER.
2. Set up the gel box for pouring by placing the black metal dams beside the gel plate in the
    appropriate slots.
3. If you will be using a stain in your agarose (ask your instructor), then wait to add the stain
    until the agarose has been heated up and has cooled to the point where you can hold your
    hand to the beaker without discomfort. Gently swirl the agarose/stain to fully mix the
    contents.
4. Slowly pour agarose gel onto gel plate, making sure that the entire surface of the gel plate is
    covered. Remove any bubbles using a stir rod.
5. Place the eight tooth white comb into the slot on the negative (black) electrode side.
6. Allow gel to cool until it solidifies (~5minutes). While waiting, hook up wires to the power
    supply. Make sure you have a micropipet, tips, and DNA samples ready.
7. Carefully remove the comb from the gel.
8. Follow the instructions for an immersion or a wick gel according to your instructor's
    directions.
Immersion gel: Add buffer into buffer reservoirs in gel box until gel is submersed. DO NOT
    POUR BUFFER DIRECTLY ONTO GEL SURFACE TO AVOID DAMAGING THE
    GEL.



                                                18
Wick gel: Cut a piece of wicking material (if not cut in advance) into two 6cm x 6cm squares.
    Place the wick on each side of the gel box assuring that the wick covers the entire width of
    the gel but only covers ~0.5cm of the surface of the gel. The other end of the wick should be
    placed such that it rests on the bottom of the buffer reservoir. Repeat with other side of gel
    box. Pour buffer into each buffer reservoir until they are full (do not allow the buffer from
    one reservoir to run underneath the gel plate and contact the other buffer). The wick material
    should absorb the buffer, so you may have to add a little more.
9. Load 20µL (unless instructed otherwise) of precut DNA sample into a well in the middle of
    the gel. Immerse tip into the well in the gel. Slowly press the control button to the second
    stop (this activates the blow-out feature assuring all the sample has been dispensed).
10. Close the gel box lid and insert the wires into their respective plugs.
11. Turn power supply on. Set voltage at 70 V. (TO AVOID ELECTRIC SHOCK, DO NOT
    TOUCH GEL BOX OR WIRES WHILE THE POWER IS ON) Record start time.
12. Observe and record any activity within the gel (gel typically requires 1-2 hours run time).
13. Your instructor will turn off the gel box if the length of your class time is insufficient.
    If your instructor has stained the gel for you, then skip to step 15.
    If your instructor does not stain the gel for you, the following procedure should be followed
    so that the DNA bands are visible.
14. Place your DNA gel into a flat dish deeper than your gel. Add enough 1X stain (as provided
    by instructor) to submerse your gel. Occasionally, gently agitate dish for 15-20 minutes.
    Stain can be reused, so return to appropriate bottle provided.
15. The next step is to destain, which removes stain from the gel while allowing it to remain
    attached to the DNA. This will enable you to more clearly see the DNA bands.
    Cover the gel with deionized water, gently agitate on occasion. Replace deionized water
    three to four times, every ten minutes over the course of 30-40 minutes.
16. Measure (in cm) the distance each band has traveled. Make a drawing of your gel, with
    distances accurately reflected. Label the number of base pairs represented by each band
    (HINT: This depends on the number of base pairs between cuts in the precut DNA sample).
    e.g. If 200 base pairs between each cut, the furthest band from the gel well would be 200
    base pairs and the next band would be 400 base pairs moving back towards the well.

Discussion Questions

1. Explain why it was beneficial to place the comb on the negative side of the gel box instead of
   the center after pouring the agarose.
2. Where would you find the smallest molecules of DNA in the gel? The largest?
3. How could this DNA sample (ladder) be used to determine the number of base pairs in an
   unknown sample?
4. Assuming you have done gel electrophoresis of dyes or proteins, explain why you think the
   DNA samples ran more slowly. (HINT: Consider molecular weight)
5. The number of base pairs in the DNA molecules that make up each band changes by the same
   amount between each band i.e. If the first band contains 123 b.p., the next band would
   contain 246 b.p., and the next band would contain 369 b.p. etc… You should have observed
   that the bands closest to the well were closer together than the bands furthest from the well.
   What conclusions about relationship between velocity and molecular weight can you draw
   from this observation?



                                                19
                                          Appendix A

Teacher instructions and suggestions for Micropipet Practice Activity

Approximate laboratory time required

45-50 minutes

Prerequisite student skills

Comprehension of metric measurement

Instructional Strategy

Most separations of proteins are on a very small scale and use micro amounts of liquids. This
requires ability to manipulate hand-held instruments called micropipets. This activity will
familiarize students with their use and offers a quick method of determining if they're using them
properly.

Materials

micropipets
plastic microfuge or vortex mixer
microcentrifuge tubes (1.5mL)
pipet tips
glycerin
distilled or de-ionized water
filter paper
food coloring dyes (red, green, blue & yellow)
6" x 6" Styrofoam sheets to act as microcentrifuge tube holders
vortex mixer or plastic toothpicks for mixing

Advance Preparation
   1. Stock dye solution preparation for 8 class sets
            a. Mix 4.0 mL desired food coloring and 4.0 mL of distilled water
            b. Add 16 uL of glycerin to mixture and mix thoroughly. (A vortex mixer is
               preferred)
            c. Divide each colored solution into 8 labeled microtubes.
   2. For each group of students prepare a kit consisting of:
            a. A Styrofoam sheet or piece of foam (microwell plates work well) to act as a micro
               test tube holder containing:



                                               20
                      4 empty microfuge tubes;
                      4 labeled microfuge tubes each with one of the four basic colors
           b. 1 micropipet
           c. filter paper




Teacher suggestions

Micropipet tips can be rinsed and reused (for this activity, other protocols require a very sterile
environment). Prepare a standard so you can compare their teal, rose, orange and chartreuse
colors. This indicates the students' ability to use the micropipets accurately.

Micropipet calibration can be verified using a good analytical balance to compare the weight of
the sample to the theoretical delivered volume. The density of water is 1g/mL or 1µg/µL so the
weight in µg should equal the volume reading specified on the micropipet. Repeating this
multiple times would increase the accuracy of the calibration.

Another method to verify accurate mixing of colored dyes by students is to use a
spectrophotometer. This method may be more appropriate for older students.




                                                 21
                                          Appendix B
Teacher instructions and suggestions for Extraction of Ink from Paper lab

Approximate laboratory time required

25 minutes

Prerequisite student skills

Precise micropipetting skills

Instructional Strategy

This activity was designed for further analysis of the ink sample, it could easily be modified to
demonstrate other concepts of solubility (see teacher suggestions below). Many situations
(including criminal investigations) involve extracting and analyzing inks or dyes from paper and
other material. This activity is a simple extraction of a water-soluble ink from a note. Analysis of
this ink sample can be done in several ways, ranging from simple inexpensive techniques to
more complex, technologically advanced methods.

Materials

Small test tube
Note (written with vis-à-vis water soluble black ink – overhead pen)
Micropipet
Distilled water
Scissors

Advance Preparation

Write out notes ahead of time for students to cut up. Heavy lettering yields a more concentrated
ink sample that will help with chromatography or gel electrophoresis.

Teacher suggestions

It would be helpful to do this activity in advance so that you know how dark a desirable ink
sample should appear (this would depend on what analysis tool you intend to use). If students
end up with too dilute of samples there are two ways to correct this. You could instruct students
to remove the letters (in the small test tube) from which the ink was extracted and add more
letters to extract more ink. Alternatively, you could instruct students to vaporize some of their
water from the water/ink extract. This could be done by heating the sample to a gentle boil or
letting it sit open to the air overnight for evaporation to occur. Any sample that disappears due
to excessive vapor removal could be rehydrated with a small sample of water.




                                                22
If the procedure in this activity is followed using a note with writing in permanent ink (non-water
soluble) or the water that acts as the solvent is replaced with ethanol (or isopropyl alcohol –
rubbing alcohol), it demonstrates that different inks or dyes are soluble only in certain solvents.




                                                23
                                          Appendix C
Teacher instructions and suggestions for Thin Film Chromatography lab

Approximate laboratory time required

20-45 minutes

Prerequisite student skills

No specialized lab skills necessary as written
Micropipetting if ink sample is from Extraction of Ink from Paper activity

Instructional Strategy

This activity was designed for further analysis of the ink sample collected from Extraction of Ink
from Paper activity. Principles of solubility and adsorption (though it may be best to leave the
explanation at solubility depending on the age group) are used to separate various colored
components from a black ink sample. This activity may be used to demonstrate solubility and
could be used in conjunction with other activities (see Gel Electrophoresis activity) that
demonstrate it as one of several mechanisms of separation.

Materials

4" diameter filter paper
250mL beaker
water or buffer
pencil
scotch tape
ruler
ink sample or ink pen

Advance Preparation

1. Verify that you have an appropriate pen (water soluble) to fit protocol.
2. Preparing a sample filter paper strip may help students follow procedure more accurately and
   reduce waste.

Teacher suggestions

While the above procedure requires only one ink sample type, one could easily incorporate other
inks (see discussion question 3) and or solvents to give students a better understanding of the
concepts of solubility and its effect upon separation. Using a water-soluble ink along with a
permanent ink in a water solvent would give students a chance to compare the two, and perhaps
come up with ideas to make the permanent ink dissolve and move upward as the water-soluble




                                                24
ink does, which may include using isopropyl alcohol – rubbing alcohol) instead of using water in
the beaker as the solvent.

When commercial dyes are run, a dozen or more bands can show up, depending on which dye is
chosen. You may want to experiment with your dyes in advance.

This procedure can be used as a stand-alone activity as written, or can be easily incorporated into
other separation techniques that include gel electrophoresis of dyes or proteins (separate due to
charge) as well as gel electrophoresis of precut DNA ladders (separation due to molecular
weight). This module ties together the above three separation techniques giving insight into how
engineers in industry might combine various techniques in order to produce an end product.




                                                25
                                          Appendix D
Teacher instructions and suggestions for Agarose Gel Electrophoresis lab

Approximate laboratory time required

30-50 minutes

Prerequisite student skills

Micropipetting if ink sample is from Extraction of Ink from Paper activity

Instructional Strategy

This activity was designed for further analysis of the ink sample collected from Extraction of Ink
from Paper activity. Principles of electrophoresis are used to separate various colored
components from a black ink sample. This activity may be used to demonstrate electrophoretic
mobility and could be used in conjunction with other activities (see Thin Film Chromatography
activity) that demonstrate it as one of several mechanisms of separation.

Materials

power supply
gel box
agarose
deionized water
1X TAE buffer
ruler
dye samples
micropipet
wick

Advance Preparation

This lab activity was written assuming the use of Horizon 58 gel boxes (borrowed from
Equipment Loan Program at Washington State University - contact (509) 335-8528. There are
other ways to make simple gel apparati using very cheap and common components - see other
papers at this website.

1. Make 1.0 L of 1X TAE buffer:
       4.84 g TRIS
       1.14 mL of 1M Acetic acid
       100 mL of .5M EDTA or .37 g EDTA (Disodium salt)
       Add deionized water bringing total volume up to 1.0 L
   Note: Each gel box requires about 125 mL of buffer to run immersion gels (less for wick
   setups).


                                                26
2. It will speed the process up significantly if the teacher has the agarose heated up and ready to
    pour (this works well on a hot plate with a stir bar).
3. If you are running this as an immersion gel, then you should add about 1-2% glycerol (by
    weight) of the total dye sample. If you are running this as a wick gel, you may want to cut the
    wicks ahead of time. Wicks can be made from filter paper or paper towels.

Teacher suggestions

While the above procedure requires only one dye sample type, one could easily incorporate other
dyes. This lab was designed to have each student actually load a gel and become familiar with
basic procedures in electrophoresis. If each student loads the same dye, any errors in technique
are likely to show up when gel is run (see question #3 in Discussion Questions). Errors could
include loading dye being injected into gel (or through it), improper micropipetting techniques,
or loading dye not entering the well. If you are looking for a more complex lab (more types of
dyes loaded see Comparison of Electrophoretic Rates of Dyes activity.


Immersion gels are easier to run if you have added the glycerol ahead of time. The glycerol (can
substitute sucrose) increases the density of the sample so that it will stay in the well instead of
diffusing into the buffer. It does create some problems (smearing of bands) when run with the
Thin Film Chromatography activity, therefore we chose to use the wicking technique (reduces
buffer volumes) as it makes for a great comparison in the Chromatography/Electrophoresis
Comparison activity that we have designed.

The wicks from a wick gel can be dried out and reused many times. Make sure that students do
not allow the buffer from one reservoir to seep under the gel plate and make contact with buffer
on the other side. This allows the electricity to flow directly through the buffer, not through the
gel, potentially affecting the electrophoretic velocity of the dyes.




                                                 27
                                          Appendix E
Teacher instructions and suggestions for Chromatography/Electophoresis Comparison

Approximate laboratory time required

50 minutes

Prerequisite student skills

Micropipetting
Explanation of how to properly load a well in a gel

Instructional Strategy

This activity was designed for further analysis of the ink sample collected from Extraction of Ink
from Paper activity. This activity demonstrates two of several mechanisms of separation.
Principles of solubility (and adsorption) and electrophoresis are used to separate various colored
components from a black ink sample. Use of a Vis-à-vis black water soluble overhead pen (as in
Extraction of Ink from Paper) yields discrepant results (conducive to INQUIRY Learning)..

Materials

4" diameter filter paper
250mL beaker
pencil
scotch tape
ruler
power supply
gel box
agarose
(1X) TAE buffer
ruler
dye samples
micropipet
wick

Advance Preparation

This lab activity was written assuming the use of Horizon 58 gel boxes (borrowed from
Equipment Loan Program at Washington State University - contact (509) 335-8528. There are
other ways to make simple gel apparati using very cheap and common components (see other
papers at this website).

1. Make 1.0 L (or more depending on number of students) of 1X TAE buffer:
      4.84 g TRIS


                                                28
        1.14 mL of 1M Acetic acid
        100 mL of .5M EDTA or .37 g EDTA (Disodium salt)
        Add deionized water bringing total volume up to 1.0 L
2. It will speed the process up significantly if the teacher has the agarose heated up and ready to
    pour (this works well on a hot plate with a stir bar).
3. You may want to cut the wicks ahead of time and demonstration of how to set this up may be
    helpful.

Teacher suggestions

While the above procedure requires only one dye sample type, one could easily incorporate other
dyes. This lab was designed to demonstrate that different separation techniques can yield very
different results. It might be a good idea to have students load more than one well.

An immersion gel technique is not used in this because the glycerol (used to increase the density
of the loading dye) would also need to be added to the dye for the chromatography procedure in
order to maintain good scientific method (for comparison sake). In the chromatography, glycerol
creates some problems (smearing of bands). Therefore we chose to use the wicking technique
(reduces buffer volumes) as it makes for a great comparison.

The wicks from a wick gel can be dried out and reused many times. Wicks can be made from
filter paper or paper towels. Make sure that students do not allow the buffer from one reservoir to
seep under the gel plate and make contact with buffer on the other side. This allows the
electricity to flow directly through the buffer, not through the gel, potentially affecting the
electrophoretic velocity of the dyes.




                                                29
                                         Appendix F
Teacher instructions and suggestions for Determining Electrophoretic Velocity

Approximate laboratory time required

45 minutes

Prerequisite student skills

Basic understanding of gel electrophoresis techniques

Instructional Strategy

This activity was designed to provide students with a mechanism to quantify electrophoretic
properties. It can be used to compare dyes, proteins, or DNA assuming all conditions are
carefully controlled. This activity can be expanded to include identifying unknowns based upon
electrophoretic velocity (see Identifying Unknowns Using Electrophoretic Velocity).

Materials

power supply
gel box
agarose
deionized water
(1X) TAE buffer
ruler
dye samples
micropipet
wick

Advance Preparation

This lab activity was written assuming the use of Horizon 58 gel boxes (borrowed from
Equipment Loan Program at Washington State University - contact (509) 335-8528. There are
other ways to make simple gel apparati using very cheap and common components (see other
papers at this website).

1. Make 1.0 L (or more depending on number of students) of 1X TAE buffer:
      4.84 g TRIS
      1.14 mL of 1M Acetic acid
      100 mL of .5M EDTA or .37 g EDTA (Disodium salt)
      Add deionized water bringing total volume up to 1.0 L

2. You should use dark colored dyes (~1% usually works - must be dark enough to leave visible
   color bands). Food coloring, Kool-Aid, colored sport drinks, candy dyes, and scientific dyes


                                              30
    and indicators all work well. Methylene blue will migrate in the opposite direction, providing
    an opportunity for inquiry learning. This lab is a simulation of protein separation. Obviously
    proteins could be used if available.
3. It will speed the process up significantly if the teacher has the agarose heated up and ready to
    pour (this works well on a hot plate with a stir bar).
4. You may want to cut the wicks ahead of time and demonstration of how to set this up may be
    helpful.

Teacher suggestions

This activity works well by itself or you could easily run a mix of dyes in one well and students
could easily look across the gel and determine what dyes are in the mix. The following activity
(Identifying Unknowns Using Electrophoretic Velocity) was designed to enhance this activity. It
tests how well students can match the conditions of the experiment from one day to another and
also how well they measured and recorded data from the previous experiment (a skill that most
students need to develop).

When dyes or proteins run in opposite directions it is because they are oppositely charged.
Negatively charged proteins will migrate toward the anode (+) and positively charged proteins
will migrate toward the cathode (-).




                                                31
                                         Appendix G
Teacher instructions and suggestions for Identifying Unknowns Using Electrophoretic Velocity
This lab must follow the previous lab (Determining Electrophoretic Velocity) so students will
have the velocities they need to identify the unknowns.

Approximate laboratory time required

45 minutes

Prerequisite student skills

Basic understanding of gel electrophoresis techniques


Instructional Strategy

This activity was designed to reinforce a previously learned skill and requires students to
maintain exact conditions to get good results. It can be used to compare dyes, proteins, or DNA
assuming all conditions are carefully controlled. By using an unknown and comparing to the
previous data, a strong emphasis is placed on accurate measurement, procedure and record
keeping.

Materials

power supply
gel box
agarose
deionized water
(1X) TAE buffer
ruler
unknown samples or mix
micropipet
wick

Advance Preparation

This lab activity was written assuming the use of Horizon 58 gel boxes (borrowed from
Equipment Loan Program at Washington State University - contact (509) 335-8528. There are
other ways to make simple gel apparati using very cheap and common components (see other
papers at this website).

1. Make 1.0 L (or more depending on number of students) of 1X TAE buffer:
      4.84 g TRIS
      1.14 mL of 1M Acetic acid
      100 mL of .5M EDTA or .37 g EDTA (Disodium salt)


                                               32
       Add deionized water bringing total volume up to 1.0 L

2. For the unknowns, you should use the same concentrations and types of dyes as used in the
    previous lab (Determining Electrophoretic Velocity).
3. It will speed the process up significantly if the teacher has the agarose heated up and ready to
    pour (this works well on a hot plate with a stir bar).
4. You may want to cut the wicks ahead of time and demonstration of how to set this up may be
    helpful.

Teacher suggestions

This activity tests how well students can match the conditions of the experiment from one day to
another and also how well they measured and recorded data from the previous experiment (a
skill that most students need to develop).

When dyes or proteins run in opposite directions it is because they are oppositely charged.
Negatively charged proteins will migrate toward the anode (+) and positively charged proteins
will migrate toward the cathode (-).




                                                33
                                        Appendix H
Teacher instructions and suggestions for Agarose Gel Electrophoresis of Precut DNA

Approximate laboratory time required

Two 45 minute sessions (depending on choice of procedure)

Prerequisite student skills

Basic understanding of gel electrophoresis techniques
Entry level knowledge of DNA

Instructional Strategy

This activity was designed to demonstrate the role of molecular weight in electrophoretic
separation. This lab introduces a third mechanism of separation when used in conjunction with
previous labs which incorporate solubility (Thin Film Chromatography) and electrophoretic
properties (Agarose Gel Electrophoresis). Although other properties also determine molecular
separation, the aforementioned are three of the most common.

Materials

power supply
gel box
agarose
Carolina BLU Gel & Buffer Stain
(1X) TAE buffer
Carolina BLU Final DNA Stain
deionized water
ruler
Precut DNA
Micropipet
wick

Advance Preparation

This lab activity was written assuming the use of Horizon 58 gel boxes (borrowed from
Equipment Loan Program at Washington State University - contact (509) 335-8528. There are
other ways to make simple gel apparati using very cheap and common components (see other
modules at this website).

1. Make 1.0 L (or more depending on number of students) of 1X TAE buffer:
      4.84 g TRIS
      1.14 mL of 1M Acetic acid
      100 mL of .5M EDTA or .37 g EDTA (Disodium salt)


                                              34
        Add deionized water bringing total volume up to 1.0 L
        (see Teacher suggestions for information about incorporating a stain into the buffer)
2. The Precut DNA (DNA ladder-123b.p.) used in this activity can be purchased (~$50 per
   classroom set) from Carolina Biological Supply (most scientific supply companies also carry
   this). To avoid potential loss or contamination of the DNA sample by students, we
   recommend preparing the DNA sample for the entire class in advance. With the above
   sample, a mix of 10 parts ladder to one part loading dye (typically comes with DNA ladder)
   is recommended for approximately 20L total loaded in each well.

3. The stain used in the procedure can be recycled, so you may wish to provide a separate
   container into which the students may place their used stain.

Teacher suggestions

There are many other sources of DNA ladders. If you purchased your ladder from a company
other than Carolina Biological Supply, we recommend following the protocols that accompany
your specific sample.
Carolina Biological Supply protocol recommends the use of Carolina BLU Stain in the agarose
gel and/or the buffer, however, decent results can be obtained via post-staining only. For precise
recommendations, we would advise you to follow the recommendations that are included with
your DNA ladder.
As an alternative to students spending a class period destaining gels, they can be fully destained
by allowing them to set overnight at room temperature in a small amount of deionized water.
Once sufficiently destained, the gel can be stored for future observation by covering in plastic
wrap or by placing in a plastic storage bag in the refrigerator. The maximum recommended
storage time is eight weeks.




                                                35

				
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