Select one of the following lesson plans:

--> Blood Analysis

--> Teacher Notes

--> Student Notes

--> Hair Analysis

--> Teacher Notes

--> Student Notes

--> Teamwork Scenario (for application of Hair/Blood Analysis)

--> DNA Fingerprinting

--> Teacher Notes

--> Student Notes

 

TEACHER INSTRUCTIONS

Goals
1. Introduce students to some of the techniques used by forensic scientists for analyzing blood.
2. Introduce students to the concept of blood type.
3. Provide opportunity for students to practice critical thinking skills in the context of scientific inquiry.

Materials
- Blood (chicken or cow; can be obtained from meat packing facility)
- Red paint
- Red food colouring
- Fresh tomato
- Fresh, raw beet
- Canned tomato sauce or spaghetti sauce
- Small plastic containers in which to aliquot each of the above substances (6 per student group)
- Set of envelopes containing cotton squares with stains from crime scene (one set per student team)
- Small dropper bottles or small plastic tubes and pipettes or droppers
- Hydrogen peroxide
- Phenolphthalein solution (instructions for prep immediately below)

Stock solution
Combine:
- 2 g phenolphthalein (powder)
- 20 g potassium hydroxide (CAUTION: caustic, strong base)
- 100 ml water.
Mix thoroughly.

Add:
- 20 g powdered zinc.
Allow 48 hours for solution to become colourless. Store in brown bottle or bottle wrapped with foil.

Working solution:
- 20 mL stock solution
- 80 mL ethanol

"Case of the Hacked High Tech Lab"
- 1 envelope with dry cotton square stained with red dye labeled "A"
- 1 envelope with dry cotton square stained with cow or chicken blood labeled "B"

NOTE: Rather than providing the students with stains to test, you may have them test the actual stains they collected from the crime scene.
- Simulated Blood Typing Kit with samples transferred to new tubes labeled "suspect 1", " suspect 2", " suspect 3", and "evidence". Place liquid from the same sample in both tubes " suspect 1" and "evidence" (e.g. Mr. Smith from the Wards kit). Place liquid from individuals with different blood types in tubes "suspect 2" and "suspect 3".

Instructions
This activity contains two parts.

Part One is intended to teach students about the catalase test for the presence of blood. While there are more sensitive tests for the presence of blood that an investigator might use, this is by far the cheapest. Following the student handout should be fairly straightforward. Students predict whether or not the substances provided will be catalase positive or negative, then they test their predictions. They also test whether each substance tests positive for blood using the phenolphthalein test. After this step they open the evidence packets provided, and test whether each stain that was found is likely to be blood.

Part Two addresses blood typing. A good way to avoid using actual human blood for this exercise is to purchase a simulated blood typing kit from a biological supply company. Their price range is approximately $35-$50.

[Back to Beginning]

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STUDENT INSTRUCTIONS

Investigators often find blood stains during their examination of a crime scene. They also find stains that could be either blood or some other similar substance, like reddish-brown paint. What other things can you think of that might look like blood? How would you test a stain to see if it is blood?

Have you ever used hydrogen peroxide to clean a cut or a scrape? What happened when the hydrogen peroxide came in contact with the blood from the wound?

Blood contains an enzyme called catalase, which breaks down hydrogen peroxide into water and oxygen gas.
2H2O2 --- catalase --> 2H2O + O2

When this reaction occurs, the oxygen gas is released as bubbles. The catalase enzyme performs an important function to living organisms because hydrogen peroxide is very toxic to living cells. Other organisms, including plants and some bacteria, also make catalase.

If you place a few drops of hydrogen peroxide on a substance that contains catalase, it will bubble profusely. These substances that bubble with the addition of hydrogen peroxide are said to test positive for catalase.

Criminal investigators do not typically use the catalase test at crime scenes. Other simple tests are better at detecting very dilute concentrations of blood – sometimes so dilute the human eye can longer see the stain. These tests (listed below), while more reliable, require more expensive chemicals.

- Benzidine
- Leucomalachite green
- Phenolphthalein
- Takayama test
- Tetra-methyl bezidine
- Luminol and Spectrophotometric tests.

Most of these tests rely on the activity of peroxidase enzymes in blood to react with a chemical stain causing it to change colour, or in the case of luminol, glow in the dark.

In this activity, you will be comparing the results of the catalase test using hydrogen peroxide with the phenolphthalein test, to see how each reacts with blood and other substances.

Which of the following substances do you think would test positive for catalase? Make a prediction for each, and explain your reasoning. Make sure you make a prediction for each substance before conducting your test.

Test each of these substances to see if it is catalase positive or negative by placing a few drops of hydrogen peroxide on a small amount of each. Record results in the table above.

SAFETY NOTE: Even though you will not be using any real human blood in this activity, you should wear appropriate protection such as gloves.

Analysis of evidence from the crime scene
Test any stains from the crime scene that you suspect may be blood stains. You should only test part of each sample and not the whole sample. Why?
Record your results below.

Which of these stains is probably blood? Could it be anything else other than blood? Check your answers against the key provided.

Once you know that a stain is blood, what else would you do as a forensic scientist? There is a lot of potential information in a blood stain.

Pattern and shape: The shape and pattern of blood drops can reveal important information about the nature of the wound from which the blood came. Was the bleeding person standing still or walking? What distance did the blood drop fall? Did the blood spatter in all directions? A good investigator would carefully photograph all blood stains from different angles both so that a forensic scientist could examine the pattern and to be able to present the evidence to a jury.

DNA: Blood contains DNA, and depending on the size of the stain and its condition (old, new, dry, etc.), a forensic scientist may be able to get enough information to obtain a highly probable match of a suspect with the evidence.
Two techniques are heavily used by forensic scientists in evaluating DNA evidence from blood or other body tissues – polymerase chain reaction (PCR) and variable number tandem repeats (VNTR’s).

Type: Blood typing can be used as an initial test to exclude some suspected sources of a bloodstain. For example, if a blood stain at the crime scene contains Type A blood, but the key suspect has Type O blood, the suspect could
be excluded as a source of the blood stain – meaning he or she definitely did not leave the blood stain. However, blood type alone usually cannot positively identify a suspect because many people share the same blood type.

Investigators have collected blood samples from each of the suspects in the case. The samples and the evidence are labeled A-D. It will be your job to type each sample. You will determine both the ABO blood type of each sample as well as the Rh factor type.

ABO blood group:
There are three alleles at the locus that determines an individual's ABO blood type, and there are four possible types --> A, B, AB, and O. Type A individuals have "A" antigens in their blood. Antigens are proteins that the body's immune
system recognizes and either mounts an immune response to, if the antigen is from a foreign source, or ignores, if the antigen is part of the body itself. Type A individuals do not mount an immune response against A antigens. If they did, the immune system would produce A antibodies that would bind to the A antigens and cause the blood to thicken and clot. Individuals who are type B don't produce antibodies against B antigens, but they do produce antibodies
against A antigens. Individuals who are type O have neither A antigens or B antigens, so they have antibodies to both types. Individuals who are type AB, have both antigens and do not have antibodies to either A or B. There are no O antigens. Type O individuals simply do not produce any antigens in this blood type group.

The Rh factor:
Another commonly tested blood antigen group is the Rh factor. Individuals who produce Rh antigens are referred to as Rh positive. Individuals who do not produce Rh antigens are referred to as Rh negative.

Follow the directions provided with your blood typing kit and determine the blood types of the samples labeled A, B, C, and D. Remember to wear gloves while handling the blood samples. Record the blood types of each individual below. Consult the key to the labels and write in the identity of each sample.

Answer the following questions regarding your results:
1.) Based on the results of the blood type analysis, can you exclude any of the suspects as having left the blood stain found at the crime scene?
2.) Based on the results of the blood type analysis, which suspect(s) could have left the blood stain at the crime scene?
3.) If you were allowed to perform additional tests using this blood stain from the crime scene, what would you recommend?

[Back to Beginning]

TEACHER INSTRUCTIONS

Goals
1. Introduce students to the thought process involved in developing a technique for forensic analysis.
2. Introduce students to the physical structure of hair.
3. Provide opportunity for students to improve skills in observation, critical thinking, and microscopy.

This activity involves two parts, which may be performed separately or as a cohesive unit. The first part requires students to examine a set of hairs. Using their observational and critical thinking skills, they will develop a procedure to identify hairs collected from crime scenes.

The second part is intended to complement any of the crime scene scenarios developed by the UCB Hughes Initiative. In this part, students examine the hairs supposedly collected from the crime scene as well as hairs from suspects and
their pets. They will use the data sheet provided to determine which suspect is the most likely match. If you intend to use both parts, it is recommended that you do them in the order described above.

Hair Analysis Activity - Part One

Materials
- Hairs from different species and individuals (humans, cats, dogs, horse, deer, rabbit, guinea pig, chinchilla, etc)
- Microscope slides
- Cover slips
- Water and droppers
- Microscopes

Instructions
Prepare a set of labeled, wet-mount slides for each group of 2-4 students. Smaller groups are probably better, but do whatever works best for your classroom. Each set should contain one slide with hairs or a single hair from each individual. Eight to ten slides total per set is a good number. A hypothetical set might include the following slides:

1.) human hair (red, long, curly)
2.) dog hair (black, straight)
3.) cat hair (grey, long)
4.) deer hair
5.) dog hair (white, wiry)
6.) cat hair (beige, short)
7.) human hair (brown, straight, short)
8.) rabbit hair
9.) human hair (blonde, wavy, long)
10.) horse hair

Instead of preparing the slides yourself, you may choose to give each team of students a set of labeled envelopes containing the hairs and ask them to prepare the wet mounts.

Instructions for preparing wet mounts of hair are included in the student handout for Part Two.

Hair Analysis Activity - Part Two
Materials
- Hairs from four different humans, one dog and one cat
- Microscope slides
- Cover slips
- Water and droppers
- Microscopes

Instructions
Prepare a set of four envelopes, labeled and filled according to specifications listed below for each team of students. Ideally, envelopes A, B, and D should contain an individual paper packet for each individual that the hair is collected
from. Each individual packet should ideally contain about 20 hairs. Some volunteers may be more amenable than others to donating this much hair.

In addition to the set of envelopes, provide each team of students with slides, coverslips, water and droppers, microscope, and copies of the student handout. Each team will need 8 copies (one per hair) of the data collection sheet.

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STUDENT INSTRUCTIONS

What would you do if you were given a collection of hairs from a crime scene and asked to determine if any of them came from the prime suspect? What characteristics of the hairs would you examine to look for differences and similarities?

Complete the following activity to help you decide how you would distinguish between hairs. At the end you will be asked to list the criteria that you would use to establish identity. There are two basic types of criteria: objective criteria (those which can be measured in units not dependent on personal judgement by the observer such as length, width, light absorbance, etc.) and subjective criteria (those which are dependent on observer judgement such as colour, texture, and shape).

Which type of criteria do think would be more reliable and more convincing? Why?

The following explanation of the physical structure of hair may be particularly useful as you examine the hairs.
Hair is composed of three principal parts:

Cuticle – outer coating composed of overlapping scales.
Medulla – central core, which may be absent.
Cortex – protein-rich structure surrounding the medulla; contains pigment.

Examine the set of labeled slides provided to you under a microscope. View each sample at both low and high power. Locate the three primary structures of each hair. As you examine the hairs, think about how they differ from one another and how you would use the differing characteristics to establish identity.

Fill in the table below and answer the following questions:

1.) How does the cuticle differ among hairs of different species? Different individuals of the same species? Try to list both objective and subjective criteria for differentiating the cuticle of different species.

2.) How does the cortex differ among hairs of different species? Different individuals of the same species? Try to list both objective and subjective criteria for differentiating the cortex of different species.

3.) How does the medulla differ among hairs of different species? Different individuals of the same species? Try to list both objective and subjective criteria for differentiating the medulla of different species.

4.) Are there other characteristics of the hairs that differ between species or individuals? List at least three. Would the criteria based on these characteristics be objective or subjective?

[Back to Beginning]

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This activity was inspired by a similar activity described in the book Crime Scene Investigations by Pam Walker and Elaine Wood, Center for Applied Research in Education, 1998.

You have been given four envelopes containing hairs from each suspect’s body and clothing as well as hairs found at the crime scene. The envelopes with hairs from the suspects are labeled with letters only (suspect A, suspect B, and
suspect C), so that you do not know the identity of the contentss. The envelope with hairs from the crime scene is labeled evidence.

Your job is to examine the samples in each envelope and compare them. If any of the samples match, it could link one of the suspects to the crime scene. Follow the steps below to complete your analysis. You may wish to split up the work within your team by having one person analyze the evidence envelope, one person envelope A, etc.

1.) Label a set of slides for each envelope with the envelope’s letter and the packet number (if there is more than one packet per envelope). The number of packets contained within the envelope will be written on the outside. You must examine each packet. Ex:

2.) Open envelope A. Open the first packet and remove one or two hairs.
3.) Measure the length of the hair in millimeters.
4.) Make a wet mount of each hair using your labeled slides.

a.) Place a small drop of water on the center of the slide.
b.) Place the appropriate hair in the drop of water so that the hair lies flat on the slide. Cut a small (1 cm) length of hair if necessary.
c.) Cover the hair and water drop with a cover slip. Ex:

5.) Examine each slide under the microscope at high power. Fill out the data sheet on each hair. You may add criteria of your own to the data sheet in the blanks provided. Refer to the handout on hair identification for help with terms.
6.) Repeat steps 2-4 with the remaining packets in each of the envelopes.
7.) Compare data sheets. Are there any packets containing hairs that appear to match hairs from the evidence envelope? Which ones? Why would you say they are a match?

Think about what an apparent match would mean in terms of evidence. How would you report your results to the district attorney or to a jury?

Hair analysis data

Label:_______________ Date:______________________________

Guide to Identification

Unfortunately, hair is not the best type of physical evidence for establishing identity. It is not possible to show with any certainty that two hairs came from the same person or animal. However, hair can be used to rule out certain suspects or scenarios. It can also be used to corroborate (support) other physical evidence if it is consistent with the rest of the evidence.How is hair analyzed?

The simplest method of identification is visual observation with the naked eye, which can indicate colour, length, and amount of curl. Hair can also be examined microscopically to reveal characteristics of its physical structure. Hair is composed of three principal parts:

Cuticle – outer coating composed of overlapping scales.
Medulla – central core, which may be absent.
Cortex – protein-rich structure surrounding the medulla; contains pigment.

The structure of hair has been compared to that of a pencil with the medulla being the lead, the cortex being the wood and the cuticle being the paint on the outside.

Cuticle: The scales of the cuticle may vary in how many there are per unit of measure, how much they overlap, their overall shape, and how much they protrude from the surface. The thickness of the cuticle may vary as well, and the
cuticles of some species' hairs may contain pigment. Characteristics of the cuticle may be important in distinguishing between hairs of different species but are often not useful in distinguishing between different people.

Medulla: The medulla may vary in thickness, continuity (one continuous structure or broken into pieces), and opacity (how much light is able to pass through it). It may also be absent. Like the cuticle, the medulla can be important for distinguishing between hairs of different species, but often does not lend much important information to the differentiation between hairs from different people.

Cortex: The cortex varies in thickness, texture, and colour and distribution of pigments. The cortex is perhaps the most important component in determining from which individual a human hair may have come. Microscopic examination can also reveal the condition and shape of the root and tip.

Biology of Hair

Hair is an outgrowth of the skin and is produced from a structure called the hair follicle. Humans develop hair follicles during fetal development, and no new follicles are produced after birth. Hair is composed of the protein keratin. Keratin is also the primary component of finger and toe nails.

Hair colour is mostly the result of pigments -- chemical compounds which reflect certain wavelengths of visible light. There are two main pigments found in human hair: eumelanin, which gives colour to brown or black hair and pheomelanin, which produces the colour in blonde or red hair. Hair colour may also be influenced by the optical effects of light reflecting and bouncing off the surfaces of the different hair layers.

Hair shape (round or oval cross-section) and texture (curly or straight) is influenced heavily by genes. However, nutritional status and intentional alteration (heat curling, "perms") can affect the physical appearance of hair.

Literature Cited:
- Fundamentals of Criminal Investigation. Charles O’Hara and Gregory O’Hara. Charles C. Thomas publisher. 1994
- The Basics of Hair. Ridgewood Dermatology & Hair Transplant Center, PC, 190 Dayton St.,Ridgewood, NJ 07450
- Crime Scene Investigations . Pam Walker and Elaine Wood. Center for Applied Research in Education -- publisher. 1998.
- Crime Scene to Court: The Essentials of Forensic Science. Peter White (ed.) The Royal Sciety of Chemistry publisher. 1998.

[Back to Beginning]

Julie Chen is computer programmer at a high-tech military research facility, which is working on new spy satellite technology. She arrived at her office early this morning and discovered her door ajar, with the glass pane broken. A quick analysis of her computer revealed that someone had used her terminal to hack into and download classified information regarding the satellite’s design. She reported the incident immediately. While Ms. Chen has a perfect record and a high security clearance, the lab’s chief researcher suspects that she may have staged the break-in and downloaded the information herself so that she could sell it to a foreign agency.

You and the other forensic specialists from the National Security Agency (NSA) have been assigned to examine the crime scene. You should look for physical evidence, document everything you find, and carefully package and label any evidence removed from the scene. You will then transport the evidence to your lab and analyze it along with samples taken from possible suspects. Please refer to the “Guide to Crime Scene Analysis” (or your preferred textbook) for specific procedures.

You will be responsible for documenting and gathering the following types of evidence from the crime scene:
Ø Fibers and/or hair
Ø Bloodstains
Ø Miscellaneous trace evidence (pieces of paper, items the thieves may havedropped, etc.)

When you arrive at the crime scene, you are told by the agent in charge that the investigators have tried to keep the area as undisturbed as possible. They inform you that the fingerprint team has already dusted the area and lifted prints, and the glass fracture pattern expert has already examined the broken window.

The NSA wants information from the physical evidence as soon as possible to answer, to the best of your knowledge, the following questions:

1.) Is there any evidence to suggest that a person other than Ms. Chen entered her office recently?
2.) Is there any evidence that would suggest that someone other than Ms. Chen has worked at her computer terminal recently?
3.) Is there any physical evidence present that could link a potential suspect to the crime or be used to exonerate Ms. Chen?

Provide a detailed written answer for each of these questions. Your supervisors may have additional questions for you later. Make sure that when you enter the crime scene, you are wearing adequate protection to prevent contaminating any evidence (gloves, booties, lab coats, hair coverings). Make sure everyone on your investigative team knows her/his role:

- team leader
- photographer and photographic log recorder
- sketch/map artists
- evidence recovery and evidence recording team

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The NSA agents have been identifying and investigating possible suspects in the unauthorised downloading of restricted high-security computer files. Your preliminary results of physical evidence from the scene have been very helpful.

As a result of your report, the agents have narrowed the suspect list to three people. They are:

Ms. Dziga Smirgov
Ms. Smirgov is the receptionist for the floor on which Ms. Chen’s office is located. She is a naturalized citizen of the United States, but the agents think she still might have relatives and friends in the former Soviet republics, who might be interested in accessing the secret information about the spy satellite. She was at her desk late the previous evening. All of the employees on this floor said either that she was still at her desk when they left the building or that they don’t remember whether she had left or not.

Dr. Anna Schneider
Dr. Schneider is a research scientist who works on a different project at the facility – non-lethal weapons development. She was seen by other employees on the floor where Ms. Chen’s office is located late in the afternoon. She claims that she dropped by to say hello to her friend Dr. Puri. Dr. Puri confirms that she did indeed drop by his office late that afternoon just before he left for home.

Ms. Belinda Jones
Ms. Jones is the facilities maintenance staff member who cleans the offices on Ms. Chen’s floor every evening after the rest of the employees go home. She claims that she did not clean the offices on Ms. Chen’s half of the floor yesterday. She rotates which offices she cleans, so that she only does half each evening. Hence, she did not notice anything suspicious. Other employees told the agents that Ms. Jones has been struggling financially.

The police have gathered the following from each suspect:
1.) Samples of their hair and the hair of their pets, if applicable.
2.) Samples of their lipstick and lip prints.
3.) Blood.
You will be asked to analyse these samples and compare them to the evidence collected from the scene. The samples from the three suspects and the evidence sample have been labeled A-D. You will not be allowed to know which suspects the letters correspond to until after you have made your conclusions.

TEACHER PREPARATION AND INSTRUCTION GUIDE FOR CAROLINA BLU STAIN

The preparation and conduct of the DNA fingerprinting laboratory is divided into the following sections:

• Preparation Of The Student Materials
• Plasmid DNA Preparation
• Restriction Endonuclease Preparation
• Migration Dye Preparation
• Preparation, Loading And Running Of An Agarose Gel For Use With Carolina Blu Stain
• Fitting the DNA Fingerprinting Exercise Into 45-Minute Periods
• Student Instructions

PREPARATION OF THE STUDENT MATERIALS

The supplies can best be provided to the class in groups of five students. The DNA samples should be kept in the refrigerator until the class is set up. At the Office of Biotechnology, for example, it is too expensive to provide DNA for every student in a class, so enough DNA, restriction endonuclease, and reaction buffer for a minimum of two groups of five students or one group of five students in every class section, is provided, whichever is greater. For the remaining groups of students, distilled water is used to replace the DNA, restriction endonuclease, and reaction buffer. Every group of students should be provided with the blue migration dye.

• 1 microcentrifuge tube (1.5 ml) containing 17 µl of a 0.025 µg/µl concentration of pBR322 DNA and labeled "C". The label should be written on the cap and body of the tube with a felt tip pen.
• 4 microcentrifuge tubes (1.5 ml), each containing 17 µl of one of four different DNA samples. The tube labeled "1" should contain 0.15 µg/µl concentration of l DNA, the tube labeled "2" should contain 0.075 µg/µl concentration of Ad-2 DNA , the tube labeled "3" should contain 0.025 µg/µl concentration of pBR322 DNA, and the tube labeled "4" should contain 0.025 µg/µl concentration of pUC19 DNA.
• 1 microcentrifuge tube (1.5 ml) containing 18 µl of a mixture of 3 µl Bgl 1 and 15 µl of reaction buffer
• The tube should be labeled "N".
• 1 microcentrifuge tube (1.5 ml) containing 40 µl of blue migration dye and labeled "D".
• 1-20 µl pipettor
• 10 sterile pipette tips of 200 µl in an appropriate container
• 1 container to hold the used pipette tips
• 5 copies of the laboratory instructions. Each group should have a letter assigned to it in the upper right hand corner of the instruction sheet.

The teacher should have available for the entire class:

• Electrophoresis gel
• Electrophoresis power supply
• 1-20 µl pipettor and 1 box of sterile 200 µl pipette tips for each gel box
• An incubator at 37° C and rack to hold the microcentrifuge tubes of the students
• 1 Sharpie marking pen
• 1 sheet of paper for each gel box that has numbered lines or boxes corresponding to the lanes on the gel.
• (Optional) Enough small, medium, large, and extra large medical gloves for the students.

Teachers should request the following supplies at least a week in advance.

• 6 boxes of pipette tips (already autoclaved)
• 34 µl of 0.025 µg/µl pBR322 per class section (minimum of 68 µl per school)
• 17µl of 0.075 µg/µl Ad-2 DNA per class section (minimum of 34 µl per school)
• 17 µl of 0.15 µg/µl DNA per class section (minimum of 34 µl per school)
• 17 µl of 0.025 µg/µl pUC19 DNA per class section. (minimum of 34 µl per school)
• 3 µl Bgl 1 per class section (minimum of 6 µl per school)
• 15 µl reaction buffer per class section (minimum of 30 µl per school)
• 144 µl Carolina Blu Gel Stain per gel (minimum of 2 gels)
• 732 µl Carolina Blu Buffer Stain per gel (minimum of 2 gels)
• 1 bottle Carolina Blu DNA Stain
• 0.7 g Agarose per gel (minimum of 2 gels)
• 70 ml 10X TBE per gel (minimum of 2 gels)
• 40 ml blue migration dye for each group of five students in all sections.
• 8 microcentrifuge tubes for each group of five students in all sections (already autoclaved)
• Fingerprinting instructions
• A note to send the unused pipette tips/boxes, 10X TBE bottles and Carolina Blu DNA Stain back to the Office of Biotechnology when finished.

[Back to the beginning]

PLASMID DNA PREPARATION

It is best if medical or dishwashing gloves are worn when preparing supplies for the laboratory to prevent the teacher's fingers from contaminating the DNA samples. The DNA will not harm the teacher, but the teacher can harm the DNA.

The DNA samples are prepared from plasmid DNA, which at the Office of Biotechnology, is provided already, at the appropriate concentration. The DNA should be kept refrigerated, except when it is being used to prepare and conduct the laboratory.

Instructions for use of the micropipettor

The 17 µl of the plasmid DNA should be put into sterile 1.5 ml microcentrifuge tubes for the students. The tubes can be prepared up to 24 hours in advance and kept in a refrigerator until the class period. The number of tubes of each type for each group of five students is described above in the PREPARATION OF THE STUDENT MATERIALS.

[Back to the beginning]

RESTRICTION ENDONUCLEASE PREPARATION

The restriction endonuclease used for the laboratory is Bgl 1, commonly referred to as Bagel 1. The endonuclease and the reaction buffer can be prepared for the students up to 24 hours in advance and kept in a refrigerator until the class period. The mixture should be prepared in separate tubes for each group of students, ie. a large amount should not be prepared and subdivided into different tubes. To obtain 18 µl of the mixture for a group of five students, 3 µl of Bgl 1 is added to a sterile 1.5 microcentrifuge tube, then 15 µl of the reaction buffer is added to the tube. To rinse the pipette tip and mix the Bgl 1 and reaction buffer, fill and unload the pipette with the sample three times. The tube should be clearly labeled as "N".

[Back to the beginning]

MIGRATION DYE PREPARATION

A blue migration dye is used to monitor the movement of the DNA during electrophoresis. At the Office of Biotechnology, such migration dye is provided ready for use. Each group of students should receive the dye, even if they are given placebos of water, instead of DNA, restriction endonuclease, and reaction buffer.

[Back to the beginning]

PREPARATION, LOADING, AND RUNNING OF AN AGAROSE GEL FOR USE WITH CAROLINA BLU STAIN

The gel can be prepared up to two days in advance of the period in which the DNA is loaded into it. If prepared in advance, 1) place the electrophoresis box in the refrigerator with the cover in place or 2) the casting tray and gel can be removed from the electrophoresis box, sealed in a plastic bag, and stored in a refrigerator.

The following description applies to the 12 cm wide x 14 cm long electrophoresis unit provided at the Office of Biotechnology, Iowa State University. The general procedures would be the same for any gel apparatus, except for the volumes of gel and buffer that are used.

1. Put on a pair of medical or dishwashing gloves and wear them throughout the procedure, including during the laboratory and the clean up. None of the chemicals used are toxic, but the gloves provide protection for persons who may have sensitive skin.
   
   
2. The electrophoresis box will come completely assembled. To prepare the box for casting a gel, followthe directions below.
   • a. To remove the lid from the box, face the box with the electrode plugs pointing to the back, and placefingers on each end of the unit while pressing thumbs against the front edge of the lid. Push thumbsagainst the lid toward the rear to disconnect the power supply leads from the plugs. Lift lid toremove from system.
   • b. Remove the gel tray by grasping each side and lifting at an angle to ease the tray out of the system (See figure 1). Rinse the combs, gel tray and gel box in distilled water to remove any residue. It is not necessary to dry the pieces.
   • c. Replace the gel tray in the system by carefully lowering the tray at an angle so the gaskets fit against the front and rear of the gel box wall (See figure 1). This should provide an efficient seal that prevents leakage of the warm agarose when poured into the tray. If the gasket has slipped out of the groove, push it back in place before lowering the tray into the box.
     
     
3. Prepare 700 ml of 1X TBE electrophoresis buffer by diluting 70 ml of 10X TBE stock solution with 630 ml of distilled water.
   
   
4. Weigh 0.7 g of agarose and pour into a 250 ml flask or beaker containing 45 ml of 1X TBE electrophoresis buffer prepared in step 3. Swirl the agarose suspension to disperse the powder.
   
   
5. Put on a heat-resistant glove. Microwave the suspension until it boils (about 30 seconds to 1 minute), swirl the flask, and alternate boiling and swirling at 15 second intervals until the solution has boiled a total of 1 minute or until there are no visible solids. Figure 1

   

6. Cool the agarose solution by adding 45 ml of the 1X TBE buffer from step 3, bringing the volume in the container to 90 ml. Swirl the solution gently to avoid trapping air bubbles.
   
   
7. Add 144 µl of Carolina Blu Gel Stain to the agarose solution. At the Office of Biotechnology, the Gel Stain is provided in a microcentrifuge tube. To get all the stain out of the tube, rinse the tube with 1X TBE buffer or distilled water and pour it in the agarose solution. Swirl the agarose solution gently until it has a uniform light blue colour.
   
   
8. Slowly pour the agarose solution into the gel tray taking care not to allow formation of any bubbles within the gel. If bubbles form, tap them with a finger until they disappear.
   
   
9. Rinse the flask or beaker immediately with plenty of tap water to prevent the agarose from hardening in it or the sink.
   
   
10. Immediately place a comb(s) of choice into the tray slot, attempting to avoid air bubble formation in the wells. Allow the gel to sit at room temperature for about 30 minutes until solid (gel will appear slightly milky).
    
    
11. After the gel is set, remove the comb(s). To remove a comb, grasp both ends of the comb and gently lift straight up with a slight back and forth rocking motion. To orient the gel tray in the running position, grasp the sides of the tray and gently lift at an angle (See figure 1). Rotate the tray 90 degrees to position the open ends toward the platinum electrodes. DNA is negatively charged and will migrate toward the positive (red) pole during electrophoresis. The wells of the gel should be nearest the negative electrode (black) end of the electrophoresis box. Carefully lower the tray into position, and secure the tray between the gel tray tabs. To verify if the gel tray is properly oriented, place the lid loosely on the box. The wells should be nearest the negative electrode (black).
    
    
12. Load the DNA samples by placing the pipette tip into the top of the well and slowly releasing the solution into the well (See figure 2). The pipettor can be kept steady by holding the barrel like a pool stick and leaning on the gel box. Put the tip into the well as vertically as possible. Do not go too deep into the well to avoid puncturing the gel. Figure 2

    

13. After the students have loaded their samples into the wells, add 732 µl of Carolina Blu Buffer Stain to the 610 ml of 1X TBE buffer that remained after step 6. At the Office of Biotechnology, the Buffer Stain is provided in a microcentrifuge tube. To get all the stain out of the tube, rinse the tube with distilled water and pour it into the buffer. Swirl the buffer until it is a uniform light blue colour.
    
    
14. Slowly fill the gel box with all the 1X TBE electrophoresis buffer to cover the gel to about a 2 mm depth. Do not pour the buffer directly on the gel.
    
    
15. Make sure the switch on the power supply is in the "Off" position before connecting the electrophoresis chamber. When ready for electrophoresis, place the lid tightly on the chamber and plug the electrical leads into the recessed output jacks of the power supply. Plug the red (+) lead into the red jack, and the black (-) lead into the black jack.
    
    
16. For operation of the power supply, follow the instructions provided with it.
    
    
17. Select the desired voltage on the power supply. A voltage of 150 will permit the electrophoresis run to be completed in about an hour. Lower voltages also can be used. The lower the voltage, the slower the DNA will migrate. For example, at a voltage of 10 the electrophoresis run will be completed in about 24 hours. The band of migration dye marks the leading edge of the DNA. The electrophoresis is complete when the leading edge of the dye has migrated 5 to 6 cm from the wells.
    
    
18. Proceed with electrophoresis: Check to be sure the blue migration dye is moving toward the positive electrode (red). If it is migrating toward the negative electrode (black), turn off the power supply, remove the lid, lift out the gel tray, turn it 180É, and repeat steps 16 and 17. CAUTION: Never remove the electrophoresis chamber lid while the power supply is on.
    
    
19. When electrophoresis is completed, turn off the power supply.
    
    
20. To remove the lid from the box, face the box with the electrode plugs pointing to the back, and place fingers on each end of the unit while pressing thumbs against the front edge of the lid. Push thumbs against the lid toward the rear to disconnect the power supply leads from the plugs. Lift the lid to remove it from the box.
    
    
21. After the electrophoresis is complete, some DNA bands will be visible. To darken the bands and make more of them visible, remove the gel tray from the electrophoresis unit and place the tray into a plastic container. Slide the gel off of the tray by pushing on one end of it. Add the Carolina Blu DNA Stain, making sure the gel is completely immersed. Do pour the stain directly on the gel.
    
    
22. Stain the gel for 15 minutes. Agitate gently, if possible.
    
    
23. Pour the stain back into the bottle. The stain can be reused 6-8 times.
    
    
24. Cover the gel with distilled water to destain. (Tap water contains chloride ions that can partially remove the stain from the DNA bands and give inferior results.) Agitate gently, if possible. During the 30 to 40 minutes of destaining, change the water every 10 minutes, if possible. During the destaining process, the bands of DNA will become more clear as the stain is removed from the remainder of the gel. It is possible to destain the gel for up to 24 hours. If the DNA bands become too light, the gel can be stained and destained again by repeating steps 21 through 24.
    
    
25. The gel can be displayed to the class by sliding it on to a piece of plexiglass and placing it on a white sheet of paper or on a white-light viewing box. Figure 3 illustrates the expected results. Dog 3 is the culprit. Figure 3

    

26. The gel can be saved for at least a month in a refrigerator by sliding it into a clear plastic bag and sealing the bag. The gel can be viewed on a white sheet of paper or a white-light viewing box without removing it from the bag.
    
    
27. None of the chemicals used in the experiment are toxic. Solutions can be poured down a conventional drain. The gel can be disposed of with other trash.
    

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FITTING THE DNA FINGERPRINTING EXPERIMENT INTO 45-MINUTE PERIODS

PERIOD 1: Teach how to use the pipettor and make the agarose gel.
After the gel is made, the teacher has two options:
a) Place the electrophoresis box in the refrigerator with the cover in place until period 2.

b) The casting tray and gel can be removed from the electrophoresis box, sealed in a plastic bag, and stored in a refrigerator until period 2. The gel can be kept in the refrigerator for up to two days before it is used.

PERIOD 2: Remove the container with the gel from the refrigerator. Conduct steps 1 (optional) through 6 of the student instructions. In step 3, the incubation at 37° C can be done for up to 45 minutes, if desired by the teacher. If the incubated DNA is not going to be loaded by the students into the gel immediately, it can be stored in a refrigerator until the migration dye is added and the gel is loaded.

After the DNA is loaded into the gel by the students, the teacher has three options:
(a) Carry out the electrophoresis.

(b) If students in another section are to load DNA into the same gel on the same day, place the cover on the electrophoresis box and store it in a refrigerator until the next class period. After the gel is loaded by the last section, add the electrophoresis buffer and carry out the electrophoresis.

(c) If another gel is to be loaded on the same day by another class section, place the cover on the electrophoresis box with the loaded gel and store it in a refrigerator until both gels can be run. After both gels are ready, add the electrophoresis buffer and carry out the electrophoresis.

After the electrophoresis is completed, there are two options for staining:
(a) Stain the gel immediately and place it in the refrigerator, as described in step 26 of the instructions.

(b) Pour enough of the electrophoresis buffer out of the electrophoresis box so that the gel is not immersed in it. Place the cover on the box and store it in the refrigerator for up to a day until it is convenient to stain it.

PERIOD 3: Stain the gel if it has not already been done, view the gel and discuss the results.

[Back to the beginning]

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STUDENT INSTRUCTIONS

DNA FINGERPRINTING

____________ (Group letter)

A farmer owned four dogs. One of the dogs chewed on her new pair of boots, which made the farmer unhappy. She wanted to pen up the culprit, but did not know which dog had done it. Fortunately, the culprit had left some strands of hair on the boot. The farmer put the hair in a plastic bag and labeled it "Hair of the culprit". She took hair from each of the four dogs and labeled the samples "Dog 1", "Dog 2", "Dog 3", and "Dog 4". She took the samples to Iowa State University and asked a scientist to determine which dog had chewed up her boots. The scientist extracted DNA from the sample of hair labeled "Hair of the culprit" and labeled it "C". The scientist extracted DNA from samples of hair from each dog and labeled them with the dog number. The teacher is providing you and your colleagues with the samples and wants you to determine which dog was the culprit. Each group of up to five students will have samples to analyse together.

Step 1. (Optional) Put on medical gloves and wear them throughout the experiment. The gloves will protect the DNA samples from contaminants that may be on your hands.

Step 2. Your group has a sample of the culprit DNA in a 1.5 ml microcentrifuge tube labeled C and samples from each of the four dogs in tubes labeled with the dog number. Keep the tubes upright throughout all the steps of the experiment to keep the DNA off the sides of the tube. Into each of the five tubes, pipette 3 µl of the restriction endonuclease Bgl l from the tube labeled N. Use a fresh pipette tip when adding Bgl 1 to each tube. To rinse the pipette tip and mix the DNA and Bgl 1, fill and unload the pipette with the sample three times. Label the five tubes with the letter assigned to your group and written in the upper right hand corner of this instruction sheet.

Step 3. Place the tubes in a rack provided by the instructor and incubate them at 37É C for 10 minutes. Bgl 1 is isolated from the bacteria Bacillus globigi. The restriction endonuclease protects the bacteria from foreign DNA, such as from a virus, by cutting it up and rendering it ineffective. The endonuclease cuts the DNA () at each site where the following sequences occur.

5'-GCCN NNN NGGC-3'

3'-CGGN NNN NCCG-5'

N can be any nucleotide, but the location and order of G (guanine) and C (cytosine) is very specific.

Step 4. Remove the tubes from the incubator and keep them upright. Into each of the five tubes, pipette 4 µl of blue dye from the tube labeled D. Use a fresh pipette tip when adding dye to each tube. To rinse the pipette tip and mix the DNA and the dye, fill and unload the pipettor with the sample three times. The blue dye is used to monitor the migration of the DNA during electrophoresis.

Step 5. Go to the electrophoresis box and record the identity of your samples before loading them on the gel. The gel has lanes on which individual samples will be run, therefore, there are numbered lines on the sheet of paper. Record on the sheet the identity of the sample that corresponds to the lane into which the sample will be loaded.

Step 6. Using a pipettor set for 20 µl, transfer your sample into the well of the appropriate lane in the gel. Place the top of the pipette tip into the top of the well and dispense the 20 µl of solution into it slowly. Do not let the pipette tip touch the bottom of the well because it will puncture the gel. Discard the pipette tip in the designated container after a sample has been put in the well and use a new one for the next sample.

The gel will be run by the instructor.

Step 7. After the gel is run, the bands on it will be viewed. From the DNA patterns on the gel, determine which of the four dogs chewed up the boots.


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Prepared by the Office of Biotechnology, Iowa State University
revised 6/94