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Extraction of ink from paper:

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

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.

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.

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.

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.

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


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.

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.


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?

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 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.

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.


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?

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


250mL beaker

pencil

scotch tape

ruler

power supply

gel box

agarose

(1X) TAE buffer

ruler

dye samples

micropipet

wick

Procedures





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.

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.


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.


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?

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





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.


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.

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).


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.





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.

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





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.




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).


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.





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.

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



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.


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.




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).


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.


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?

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:

· 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.

Appendix B

Teacher instructions and suggestions for Extraction of Ink from Paper lab


25 minutes


Precise micropipetting skills


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.

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.

Appendix C

Teacher instructions and suggestions for Thin Film Chromatography lab


20-45 minutes


No specialized lab skills necessary as written

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


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


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 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.

Appendix D

Teacher instructions and suggestions for Agarose Gel Electrophoresis lab


30-50 minutes


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


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).

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.

Appendix E

Teacher instructions and suggestions for Chromatography/Electophoresis Comparison


50 minutes


Micropipetting

Explanation of how to properly load a well in a gel


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


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:

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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.

Appendix F

Teacher instructions and suggestions for Determining Electrophoretic Velocity


45 minutes


Basic understanding of gel electrophoresis techniques


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



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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 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.



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 (-).

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.


45 minutes




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



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2. For the unknowns, you should use the same concentrations and types of dyes as used in the previous lab (Determining Electrophoretic Velocity).



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 (-).

Appendix H

Teacher instructions and suggestions for Agarose Gel Electrophoresis of Precut DNA


Two 45 minute sessions (depending on choice of procedure)



Entry level knowledge of DNA


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).


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(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.

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