What is DNA Fingerprinting? Case of the Bloody Micropipettor- Teacher Guide

How is DNA evidence prepared and analyzed in a crime case? Students perform agarose gel electrophoresis to analyze DNA samples from a mock crime scene. Based on DNA fingerprinting profiles that simulate samples (the DNA source is actually a bacterial virus) from the two suspects, the victim, and the blood on the murder weapon, students determine which suspect likely committed the crime. This activity helps students understand how DNA variation in individuals can be analyzed in practical applications such as genetic testing and forensics.

Classroom time needed for this lab

• two class periods (~50 minutes each)

You will need to prepare the following materials (recipes to follow):

• 1X TAE
• 0.8% agarose in 1X TAE
• Crime scene DNA samples
• 0.025% methylene blue solution

You will need the following equipment and supplies:

• Micropipets and tips to load dye samples
• Small microcentrifuge tubes (0.5 ml or 0.65 ml size)
• Electrophoresis units and power supplies
• 0.8% agarose in 1X TAE (liquid)
• 1X TAE for electrophoresis units
• Microwave oven
• Hot water bath for keeping agarose liquified

Setting up the classroom:

Distribute at each lab station (we use four students per group):

Day 1:

• A microtube of each DNA sample (1, 2, 3, and X). Each microtube contains 10 ul of the DNA sample.
• Micropipet and tips
• Electrophoresis unit and power supply (Label each electrophoresis unit with a number written on a piece of tape.)

Day 2:

• Small container, such as Rubbermaid, to use as a methylene blue staining tray
• Methylene blue solution
• Light boxes

We use a central station for agarose and 1X TAE. We use a hot water bath to hold four small bottles, each containing 125 ml of 0.8% agarose. The high temperature keeps the agarose liquified. At room temperature we have four large bottles, each containing 1000 ml of 1X TAE.

If necessary, demonstrate to students how to use the micropipet.

Be sure the students record the number of their electrophoresis unit on their lab handout. This will help students find their agarose gel during the next class.

When students stain their gels with methylene blue, they need to work carefully. The methylene blue can stain their clothes and skin. During destaining, the water rinse can be disposed in the sink.

Day 1:

After the electrophoresis units complete their run, turn off the power supply and store the gels in the refrigerator when time permits. The gels can sit in the 1X TAE for awhile. After the gels are wrapped in saran wrap and stored in the refrigerator (be sure to put masking tape on the saran wrap and record the electrophoresis unit number), the 1X TAE in the electrophoresis units can be poured back into the large bottles for reuse. Rinse the electrophoresis units with water. The units should have a final rinse with deionized or distilled water before being put away for a long period of time.

Day 2:

The used gels can be disposed or taken home by the students. If the students want the gels, cover them with saran wrap.

The methylene blue solution can be reused up to five times.

1. We use the Micropipets available from Life Technologies, and we use regular yellow tips (1-200 ul). These micropipets are much less expensive than micropipets commonly found in research laboratories, and they are more likely to survive in student hands.
   
   
2. Be sure to calculate how much agarose you will need for the lab. You can add a defined volume of water into the gel tray to determine the volume of agarose that would be needed for each tray.
   
   
3. Be sure to calculate how much 1X TAE you will need for the lab. Using a graduated cylinder, you can pour a defined volume of water into the electrophoresis unit to determine the volume of 1X TAE that would be needed for each unit.
   
   

Recipes

1X TAE Buffer

TAE is the buffer used in gel electrophoresis.

Procedure

1. Put 48.4 g Tris base, 11.4 ml glacial acetic acid, and 20 ml of 0.5 M EDTA into a 1000 ml volumetric flask or graduated cylinder.
   
   
2. Add distilled water to make a total volume of 1 liter. This forms a 10X stock solution.
   
   
3. Dilute the 10X stock solition to make a 1X working solution (100 ml 10X stock to 900 ml distilled water).
   
   
4. Store 1X TAE buffer solution in a large container. The 1X TAE can be kept indefinitely at room temperature. This buffer can be reused several times before disposing down the sink.
   
   

0.8% Agarose

Agarose is the gel matrix used separating molecules, such as DNA or dyes, during electrophoresis.

1. Weigh 1 gram of agarose on a folded piece of weighing paper and add to empty 250 ml bottle or flask.
   
   
2. Add 125 ml of 1X TAE buffer solution to agarose. Note: The container should never be filled more than half-way in order to prevent the solution from boiling over.
   
   
3. If a bottle is used, cap the bottle loosely to release air during boiling. If you using a flask, cover the opening and neck of flask with plastic wrap.
   
   
4. Mix solution by swirling. Microwave the agarose solution at high heat until powder is completely dissolved. The length of time required will vary depending on the microwave oven. The molten agarose solution should look clear.
   
   
5. To keep the agarose liquified (for example, during several biology classes), store the bottle or flask of agarose in a hot water bath between 600 and 700C. Be sure that the bottle or flask is covered to prevent evaporation. A hot plate with a pot of water can substitute for a laboratory water bath.
   
   
6. If there is agarose left over in the container, you can let the agarose solidify and store it at room temperature until next use. Be sure that the container is well covered.
   
   

6X Loading Dye

This concentrated dye mixture is added to the DNA samples to help in loading the gels. The dyes migrate through the gel during the electrophoresis run so that you can judge how the samples have traveled.

The dye mixture is also used to prepare the practice samples for handling the micropipets.

Prepare 2% bromophenol blue stock solution:

1. Measure 0.2 g bromophenol blue powder on a square piece of folded weighing paper.
   
   
2. Using the crease in the paper, pour the dye powder into a tube. The tube should be able to hold between 10 and 15 ml.
   
   
3. Add 10 ml deionized or distilled water to the dye and cap the tube. Shake the tube well to dissolve the dye in the water. The powder may not completely dissolve in water - be sure to shake the solution well before using.
   
   
4. The 2% dye stock be stored indefinitely at room temperature.
   
   

Prepare 2% xylene cyanol stock solution:

1. Measure 0.2 g xylene cyanol powder on a square piece of folded weighing paper.
   
   
2. Using the crease in the paper, pour the dye powder into a tube. The tube should be able to hold between 10 and 15 ml.
   
   
3. Add 10 ml deionized or distilled water to the dye and cap the tube. Shake the tube well to dissolve the dye in the water. The powder may not completely dissolve in water - be sure to shake the solution well before using
   
   
4. The 2% dye stock be stored indefinitely at room temperature.
   
   

Prepare 50% glycerol solution

1. Pour 10 ml glycerol into a 25 ml graduated cylinder (note: avoid using a pipet since glycerol is very viscous). Next, add 10 ml deionized or distilled water to the same cylinder. Use a stirring rod (a pipet would also work) to mix the glycerol and water. The glycerol solution can be stored in a small, closed container in the refrigerator.
   
   

Prepare 6X Loading dye

1. In a small tube with a cap, mix the following ingredients:

   6.0 ml	50% glycerol
   1.0 ml	2% bromophenol blue solution
   1.0 ml	2% xylene cyanole solution
   2.0 ml	deionized or distilled water

2. The 6X loading dye solution can be stored indefinitely in the refrigerator.
3. 
   

Crime scene DNA samples

The DNA samples used in the lab consist of bacteriophage lambda (l) DNA digested with different enzymes. Each restriction enzyme digestion can be divided into about 30 samples. This is enough for 60 students working in groups.

Set up the restriction enzyme digests as shown below (listed in order of addition):

Sample #1: Lambda EcoRI digestion

Incubate DNA samples at 37C for at least 2 hours and up to 12 hours. Add 80 ul 6X loading dye to the digested DNA. The DNA sample can be stored indefinitely in a refrigerator.

Sample #2: Lambda Pst I digestion

Incubate DNA samples at 37C for at least 2 hours and up to 12 hrs. Add 80 ul 6X loading dye to the digested DNA. The DNA sample can be stored indefinitely in a refrigerator.

Sample #3: Lambda Hind III digestion

Incubate DNA samples at 37⁰C for 2 hours. Add 80 ul 6X loading dye to the digested DNA. The DNA sample can be stored indefinitely in a refrigerator.

Sample #X: Lambda Hind III + l Pst I

This sample is a mixture of two separately digested DNAs, Lambda Hind III and Lambda Pst I. After the DNAs are digested, they are combined into one sample.

Lambda Hind III

Lambda DNA	80 ul
10X React 2	20 ul
Deionized or distilled water	80 ul
Hind III	20 ul
Total	200 ul

Lambda Pst I

Incubate DNA samples at 37⁰C for 2 hours. Add 40 ul 6X Loading dye to each tube. After adding loading dye, combine the lambda Hind III and lambda Pst I reactions into one tube.

Before the lab, label small microtubes (0.5 or 0.65 ml size) with "1," "2," "3," or "4." Prepare enough tubes for all the lab stations. In our labs, the students work in groups of four and use two of each sample. In a class of 28 students, 14 samples each of 1-4 would be prepared.

Before classes start, distribute the restriction enzyme digestions into the proper microtubes. Each tube gets 12 ul of the DNA sample, enough for a single well. Make sure each restriction enzyme digestion is added to correct set of numbered tubes. Store samples in refrigerator until needed.

0.025% Methylene blue staining solution

1. Weigh 1 g of methylene blue trihydrate powder (m.w. 373.9) in 100 ml deionized or distilled water. Stir until powder is dissolved in water. This is a 1% methylene blue concentrate, which can be stored indefinitely at room temperature.
   
   
2. Add 10 ml 1% methylene blue concentrate to 390 ml deionized or distilled water. Stir until methylene blue is evenly mixed in solution. This solution is 0.025% methylene blue, which can be stored indefinitely at room temperature.
   
   
3. The methylene blue solution can be reused four or five times.
   
   

Sources of laboratory materials

Item	Supplier and catalog no.
Agarose	Life Technologies 15510-019
Genemate yellow 1-200 ul pipet tips	ISC P-3200-1
Graduate micropipets, 5/pack	Life Technologies 10245-066
Horizon 58 horizontal gel electrophoresis apparatus	Life Technologies 41060-013
Life Technologies Electrophoresis Power Supply	Carolina P7-21-3700
Microfuge tubes, 0.5 ml, 1000 per box	VWR 20170-310
Microfuge tubes, 1.7 ml, 500/box	VWR 20170-331
*Precision utility water bath	VWR 13470-030
Corning hot plate/stirrer	VWR 33920-219
White light transilluminator and AC adaptors	Carolina K3-21-6214, K3-216216
Tris base, 1kg	VWR JT4099-2
Boric acid. 500 g	VWR JT4035-1
EDTA, disodium salt, dihydrate, 100 g	Life Technologies 15576-010
Ohaus balance, CT series 200g	VWR 11378-059
Glycerol, 500 ml	Sigma G7893
Bromophenol blue	Sigma B5525
Xylene Cyanole	Sigma X4126
Bacteriophage Lambda DNA, 500 ug	Life Technologies 25250-010
EcoRI restriction enzyme, 5000 units	Life Technologies 15202-013
Hind III restriction enzyme, 5000 units	Life Technologies 15207-012
Pst I restriction enzyme, 3000 units	Life Technologies 15215-015
Methylene blue trihydrate powder, 25 g	Sigma M9140

*Hot plates can be substituted

DATASHEET

Link to the Worksheet/Datasheet for the DNA Fingerprinting lab

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BIOTECH Project Department of Molecular and Cellular Biology The University of Arizona Designed by: Ken Kubo, Ph.D. September 15, 2000 Last Modified March 1, 2002 Nadja Anderson, Ph.D. nadja@email.arizona.edu http://biotech.biology.arizona.edu