Materials Included In Kit

Additional Materials Required

Prelab Preparation

Preparation of 1X Electrophoresis Buffer

1. Measure 20 mL of 50X TAE buffer in the graduated cylinder.
2. Add the 50X buffer to 980 mL of distilled water in a 1000-mL Erlenmeyer flask.
3. Mix with a glass stirring rod.
4. Seal with Parafilm M^® or plastic wrap.
5. Label and store in a refrigerator.
6. Repeat.

Note: Prepare enough buffer solution to allow each group to cover the gel in the chamber to a depth of about 2 mm. Depending on the type of electrophoresis units being used; the amount of buffer needed could be as much as 300 mL per chamber. The gel preparation requires an additional 60 mL of buffer to make a 6 x 6 cm gel.

Make fresh buffer weekly.

Preparation of 1X Methylene Blue Electrophoresis Stain

1. Measure 120 mL of 10X methylene blue staining solution in the graduated cylinder.
2. Add the staining solution to 1080 mL of DI water in a 2-L beaker.
3. Mix with a glass stirring rod.
4. Seal with Parafilm M^® or plastic wrap.
5. Label and store in a refrigerator.

Note: 40 mL is enough to stain a gel in the staining tray that is provided.

Safety Precautions

Electrical Hazard: Treat these units like any other electrical source—very carefully! Be sure all connecting wires, terminals and work surfaces are dry before using the electrophoresis units. Do not try to open the lid of the unit while the power is on. Exercise extreme caution in handling the methylene blue—it will readily stain clothing and skin. Wearing chemical splash goggles and gloves is strongly recommended. Wash hands thoroughly with soap and water before leaving the laboratory. Please consult current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. All solutions used in this lab may be disposed of down the drain using copious amounts of water according to Flinn Suggested Disposal Method #26b. Used gels may be disposed of in the regular trash according to Flinn Suggested Disposal Method #26a. The DNA in this kit is derived from bacteriophage samples. It is not pathogenic to humans and therefore it is not considered a biohazard.

Lab Hints

Preparation

• Store DNA samples in the refrigerator until ready to use. DNA stored at room temperature or warmer may degrade over time. Short periods at room temperature will not affect results.
• Copy the Police Report for each group of students.
• The gel preparation pages have been listed separately so that they may be copied for student use if desired.
• Gel preparation requires approximately 10–20 minutes plus at least an additional 20 minutes for the gel to solidify (60 minutes is optimal solidification time). Longer solidification times “harden” the gel, minimizing tears and creating more distinctive bands of DNA.
• When preparing agarose gels using a stirring hot plate, rotate a magnetic stir bar very slowly to diminish the number of bubbles in the agarose solution.
• Without a balance, prepare all six 0.8% gels simultaneously. Measure 375 mL of electrophoresis buffer in a 500-mL Erlenmeyer flask, add the entire 3 g of agarose and dissolve as directed.
• The concentrated Methylene Blue Electrophoresis Stain, concentrated Electrophoresis Buffer and Agarose Solution may be stored at room temperature.

Procedure

• Have students practice pipetting 10 μl of tap water into a defective gel while waiting their turn to load the gel. Another alternative would be to prepare practice gels with less expensive agar instead of agarose. The Pipetting Practice Kit, Flinn Scientific Catalog Number FB1649, is a reusable, more durable option that works very well.
• Run the gel at 5 V/cm. For example, if the electrodes are 25 cm apart then the gel should be run at 125V. Running the gel at a higher voltage may cause the agarose to melt, hindering its ability to act like a molecular sieve. Run a practice gel to determine the length of time the gel will need to run. 20–120 minutes is a reasonable range to expect results. In general, longer electrophoresis runs at lower voltages will increase the resolution of the resulting bands. If necessary, connect the power source to a household automatic timer to end the sample run.
• The sample data included with the kit were collected after 42 minutes at 125 V using an Edvotek M-12 electrophoresis apparatus.
• Gel samples submerged under buffer may be stored in the refrigerator for up to two weeks.
• The DNA samples contain bacteriophage DNA fragments, bromphenol blue tracking dye, xylene cyanole tracking dye, sucrose and TAE buffer.
• Sucrose is added to a DNA sample to make the DNA sample denser than the TAE buffer. This causes the DNA sample to sink into the sample well in the gel.
• Bromphenol blue migrates at the same rate as a 200 – 400bp DNA fragment, toward the beginning of the sample run.
• Xylene cyanole migrates at the same rate as a 4000bp DNA fragment. It helps visualize the middle to end of the sample run.
• TBE buffer may be used in place of the TAE buffer included with this laboratory kit.
• A 10 μL micropipet with disposable tips may be used instead of the disposable needle-tip pipets.

Teacher Tips

• Enough materials are provided in this Super Value Kit for 5 classes each with six groups of students. This laboratory activity can reasonably be completed in two or three 50-minute class periods. If the gels have been prepoured and are ready to use, electrophoresis setup, sample transfer and the start of electrophoresis will take approximately 50 minutes. The electrophoresis of samples will take 20 minutes to 2 hours depending on the voltage and equipment used to run the samples. Staining and sample analysis will require an additional 50-minute period.
• Extend the lesson by incorporating a historical or new technology research project prior to the laboratory.
• Extend the lesson by role-playing the job of a forensic technician sampling a crime scene for “DNA.”
• Extend the lesson by extracting DNA from wheat germ, strawberries or cheek cells (see Flinn Scientific Kit FB1562).

Answers to Questions

1. Explain the function of each of following components of gel electrophoresis:
   1. Agarose gel—The agarose gel is the microscopic “strainer” that separates big molecules from small ones because of the size of the pores within the agarose.
   2. Electrophoresis buffer—It is the liquid conductor that carries the electric current.
   3. Wells in the gel—They hold the sample that will travel through the gel.
   4. Electric current—It provides the force that moves the sample through the gel.
2. List one important safety precaution that must be followed when performing any type of gel electrophoresis.

   Ensure that the work surface is dry, connecting wires and terminals are dry, do not open the electrophoresis chamber while the power is on, wear chemical splash goggles, chemical-resistant gloves and apron.

Analysis 

1. Using a metric ruler, measure the migration distance in millimeters for each band and sketch the observed DNA banding pattern on the DNA Banding Worksheet.

   DNA Banding Worksheet

   {11160_Answers_Figure_1}
2. Complete Data Table 1.

   Data Table 1

   {11160_Answers_Table_1}

Post-Lab Analysis and Conclusions 

1. Evaluate the resulting banding pattern of the simulated crime scene DNA and possible suspects: Give your opinion as to the identity of the guilty individual. Justify your opinion.

   DNA Sample 3 and DNA Sample 4 match. Zack Jensen, the curator, is the art thief.

2. List three errors that could affect the outcome of any gel electrophoresis procedure.
   1. Not placing the sample deep enough into the well or not placing enough sample into the well.
   2. Puncturing the well with the pipet tip causing the dye to actually run below the gel.
   3. Connecting the wires to the power supply or chamber incorrectly.
   4. Contaminating pure samples by using the same pipet tip in different samples.
   5. Not recording which sample went into which well.
3. Briefly summarize how gel electrophoresis is used to separate molecules.

   Student answers will vary but should include how this technique involves the use of a gelatin-like material that acts as molecular filter paper. When samples are placed in wells within the gel and the electricity turned on, the resulting electric field causes the molecules, which make up the samples, to separate according to their charge, size and shape.

4. Why would a forensic scientist use the Polymerase Chain Reaction technique to prepare DNA samples for analysis?

   Student answers will vary but should include detail about the need to amplify the amount of DNA present in very small DNA samples that might be present at a crime scene.

Teacher Handouts

11160_Teacher1.pdf

Introduction

How can a mixture of biochemicals, too small to be seen with even a high-powered microscope, be separated from one another? This was a dilemma facing scientists until the development of a process that has now become standard in many laboratories worldwide—gel electrophoresis. Laboratories rely heavily on this proven and reliable technique for separating a wide variety of samples, from DNA used in forensics and for mapping genes, to proteins useful in determining evolutionary relationships.

Concepts

• Gel electrophoresis
• Restriction fragment length polymorphism (RFLP)
• Genes
• Variable number of tandem repeats (VNTR)
• Polymerase chain reaction (PCR)

Background

Gel electrophoresis is a laboratory technique used to separate segments of deoxyribonucleic acid (DNA) or proteins according to the size of the segment and the relative electric charge of that segment. In 1950, a scientist named Oliver Smithies (born 1925) determined that a gel made of starch acts like a molecular filter or sieve for proteins when it is positioned between positive and negative electrodes. Dr. Smithies discovered that proteins with different sizes, shapes and molecular charge move through the gel at different rates with small fragments moving faster through the maze of microscopic pores toward the electrode with the opposite charge (see Figure 1). For example, a negatively charged protein migrates through a gel toward the positive electrode which is called an anode. DNA sequencing methods are based upon Dr. Smithies’ protein electrophoresis methodologies and principles.

DNA fingerprinting methods were first published in 1984 by Sir Alec Jeffries (born 1950). Sir Jeffries studied inherited variations within genes to determine the cause of specific inherited diseases. Sir Jeffries discovered that certain enzymes cut the DNA sequence at specific points. When these samples were analyzed using gel electrophoresis, a unique pattern was produced. These unique DNA fingerprints result from the variation in DNA created by mutation and cross-over during parental meiosis. Specifically the variation is created by changes in the number of repeating DNA base-pair sequences between genes on the chromosomes. Every living thing, except identical twins or asexual offspring, has a unique number of tandem DNA repeats called variable number of tandem repeats (VNTR). Since these tandem repeats are not part of a gene, they do not affect the viability of the organism. Sir Jeffries’ methods have been slightly modified and refined for use in DNA sequencing laboratories across the world today.

In order to determine the DNA sequence of any living thing, the DNA must first be extracted from the organism’s nucleus. This means that any cells that contain a nucleus can be used to identify the organism’s DNA sequence. The sample of cells is homogenized in a blender or crushed in a mortar and pestle with a salt compound and a detergent containing sodium dodecyl sulfate (SDS) to break the cell membranes and expose the DNA. An enzyme is added to the mixture to facilitate the uncoupling of the DNA from its histones (DNA proteins). When alcohol is added on top of the cell mixture, the DNA will move into the alcohol layer due to solubility and can then be easily retrieved.

At this point the DNA strands are too long to run in a gel so the DNA must first be fragmented at very specific base-pair locations by a restriction enzyme. Enzymes that break DNA molecules at internal positions are called restriction endonucleases. Enzymes that degrade DNA by digesting the molecule from the ends of the DNA strand are termed exonucleases. There are several different restriction enzymes available to molecular biologists. Each restriction enzyme recognizes a specific nucleotide sequence. The enzyme “scans” the length of the DNA molecule and then digests it (breaks it apart) at or near a particular recognition sequence. The specific sequence may be five to sixteen base-pairs long. For example, the EcoRI endonuclease has the following six-base-pair recognition sequence: EcoRI breaks the DNA at the locations indicated by the dotted line and produces “ragged-ended” sequences, often called sticky ends. Other endonucleases that cut the DNA cleanly at one specific base-pair produce what are called blunt ends.

Restriction endonucleases are named using the following convention:

EcoRI

E = genus Esherichia
co = species coli
R = strain RY13
I = first restriction enzyme to be isolated

The first letter (capitalized) indicates the genus of the organism from which the enzyme was isolated. The second and third letters (lower case) indicate the species. The additional letters indicate the particular strain used to produce the enzyme. The Roman numeral denotes the sequence in which the enzymes from the particular species and strain of bacteria have been isolated.

Since the average human DNA sequence is more than 3.2 billion base-pairs long (30,000 genes), there may be as many as 750,000 fragments of DNA after a single restriction enzyme completes the fragmentation of a single cell’s DNA. This process, called restriction fragment length polymorphism (RFLP), occurs because of each organism’s unique VNTR sequence discovered by Sir Jeffries.

The RFLP fragments, created by the restriction enzyme, are loaded into wells made in an agarose gel. Agarose is a refined form of agar which is made from seaweed. The agarose gel is positioned between two electrodes with the wells toward the cathode (negative electrode). This allows the negatively charged DNA to move toward the anode (positive electrode). The electrophoresis chamber is filled with a buffer solution, bathing the gel in a solution that shields the system from changes in pH.

The DNA fragments are white to colorless and appear invisible in the gel. Molecular biologists add colored tracking dyes to monitor the progress of the sample moving through the gel. Typically, two dyes are added, one that migrates at a rate similar to the smaller DNA fragments and one that migrates at a rate similar to the largest DNA fragments. Once the first dye migrates to within 1 cm of the end of the gel, the power is shut off to the electrophoresis chamber. The DNA stops migrating since the electromotive force stops.

The agarose gel is then carefully removed from the electrophoresis chamber and transferred into a staining tray. The stain binds to the DNA fragments revealing a banding pattern. Molecular biologists use a radioactive stain and X-ray film to visualize the banding pattern. In this experiment a colored dye that may be viewed with white light is used. The banding pattern is unique since the DNA sample is unique to each individual or organism, except identical twins or asexual offspring of less complex organisms. The banding pattern is measured against a series of known DNA standards and samples prepared and analyzed with the unknown DNA sample. For example, paternity test runs include a known standard of human DNA plus samples of the mother, likely fathers, and that of the child. A match is determined by calculating the probability of an individual having a particular combination of bands in a population. The more matching bands that are observed in the electrophoresis gel, the more likely it is that the DNA comes from the same person.

A second sample may be run using a different restriction enzyme. A different banding pattern will be revealed for the same DNA samples because the DNA sequence is cleaved at a different base-pair location. The probability of two individuals having identical banding patterns for two different DNA cuts tends to be greater than the current human population with the exception of identical twins who share identical genotypes. The theoretical risk of a coincidental match is 1 in 100 billion.

The mobility of the negatively charged DNA fragments in an electrophoresis chamber may be standardized by running the fragments repeatedly under identical conditions (e.g., pH, voltage, time, gel type, gel concentration). Under identical conditions, identical length DNA fragments will move the same distance in a gel. Thus, the size of an unknown DNA fragment can be determined by comparing distance on an agarose gel with that of DNA marker samples of known size. The size of the DNA fragment is usually given in nucleotide base-pairs (bp). The smaller the DNA fragment, the faster it will move through the gel during electrophoresis. Another way to look at this is—smaller segments of DNA are closer to the anode (+) end of the electrophoresis chamber and farther away from the wells where they were loaded.

As always, good scientific protocol is critical to the outcome of any laboratory analysis. Sloppy work might convict the wrong person or let a guilty suspect go free. Consequently, analysts must carefully document which restriction enzyme was used, the conditions and chemicals that were used, and the names of all known standards and controls that were prepared with the DNA sample.

RFLP is limited by the quantity of DNA available and the degree of degradation of the sample. A newer technique being utilized for forensic testing is known as the Polymerase Chain Reaction (PCR). This technology can be used to amplify (make numerous exact duplicates of) as little as a single molecule of DNA. The amplified sample may then be digested by restriction enzymes, electrophoresed, stained, and analyzed. PCR technology is able to analyze any detectable DNA sample, no matter how small or degraded.

Experiment Overview

The purpose of this activity is to demonstrate the separation technique known as gel electrophoresis. This process will be used to identify DNA samples from a simulated crime scene, along with samples from several “suspects.”

Materials

Safety Precautions

Be sure all connecting wires, terminals and work surfaces are dry before using the electrophoresis units. Electrical Hazard: Treat these units like any other electrical source—very carefully! Do not try to open the lid of the unit while the power is on. Use heat protective gloves and eye protection when handling hot liquids. Wear chemical splash goggles, chemical-resistant gloves and apron. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure

Part A. Loading a Gel

1. Assemble the electrophoresis unit according to the teacher’s instructions.
2. Place the electrophoresis unit in a horizontal position on top of a piece of white paper on a level table or countertop. Do not move the unit after loading the samples.
3. Gently slide a gel from a resealable bag into the casting tray with the wells toward the cathode (–) end of the unit.
4. Carefully position the gel and tray into the electrophoresis chamber. Caution: Be careful not to break or crack the gel. If the gel is damaged it should not be used as the breaks and cracks will affect the results.
5. Pour enough electrophoresis buffer into the unit to submerge the entire gel surface to a depth of 2–5 mm. If the gel begins to float away, reposition it on the tray.
6. By convention, DNA gels are read from left to right, with the wells located at the top of the gel. With the gel lined up in the electrophoresis chamber and the wells to the left, load the contents of DNA Sample 1 into the well closest to you. Consequently, when the gel is turned so that the wells are at the top, “1” will be in the upper left corner. If there will be empty wells in the gel, leave the outside (end) wells empty, since they are most likely to give aberrant results.
7. Place the DNA Banding Worksheet on the counter in the same orientation as the electrophoresis unit. The small rectangles on the paper correspond to the wells in the gel (see Figure 1 in the Background section).
8. Shake the DNA sample microcentrifuge tubes and lightly tap the bottom of each tube on the tabletop to mix the contents.
9. Withdraw 10 μL of DNA Sample 1 from microcentrifuge tube by filling only the needle tip of a clean pipet. Note: Fill the tip by squeezing the pipet just above the tip, not the bulb. Be careful not to draw the sample further up the pipet (see Figure 2).
   {11160_Procedure_Figure_2}
10. Dispense the sample into the first well by holding the pipet tip just inside the well. The sample will sink to the bottom of the well. Caution: Do not puncture the bottom or sides of the well. Do not draw liquid back into the pipette after dispensing the sample (see Figure 3).
    {11160_Procedure_Figure_3}
11. Record the sample name on the DNA Banding Worksheet in the appropriate well box.
12. Using a fresh pipet, withdraw 10 μL of DNA Sample 2 and load it into well 2, adjacent to DNA Sample 1.
13. Record the sample name on the DNA Banding Worksheet in the appropriate well box.
14. Repeat steps 12 and 13 for the remaining DNA samples. Use a clean pipet for each sample. Load each sample into the adjacent well. Each student group will load 4 wells.

Part B. Running a Gel

1. Place the lid on the electrophoresis chamber and connect the unit to the power supply according to the teacher’s instructions.
2. Run the gel as directed by your teacher. Note: Bubbles should form along the electrodes in the chamber while the sample is running. The bubbles are the result of the electrolytic decomposition of water—hydrogen at the cathode and oxygen at the anode.
3. Turn off the apparatus to stop the gel when the first tracking dye is 1 cm from the positive end of the gel. (This may take 30 minutes to 2 hours.) The time necessary to run a gel depends on the type of electrophoresis apparatus and the applied voltage.
4. When the power is off, remove the cover and carefully remove the gel tray from the chamber. Place the gel tray on a piece of paper towel. Note: Be careful not to break or crack the gel.

Part C. Staining the Gel
For best results, stain the gel immediately, destain and then place in a refrigerator overnight.

1. Slide the gel off the tray and into the staining tray. Note: Do not stain the gel tray.
2. Gently pour 40 mL of the methylene blue electrophoresis staining solution into the staining tray.
3. Allow the gel to stain for 5–10 minutes.
4. Pour off the stain into a glass beaker. The stain may be reused. Be careful not to damage the gel.
5. To destain the gel, gently pour room temperature distilled water into the staining container. Note: Do not exceed 37 °C, warmer water may soften the gel.
6. Occasionally agitate the water for 10 minutes.
7. Pour off the water into a waste beaker.
8. Repeat steps 5–7 until the DNA bands are distinctly visible.
9. If the bands become too light, repeat steps 2–8.

Part D. Storing the Gel
Stained gels may be stored in a laboratory refrigerator for several weeks.

1. Label a resealable bag with the group name and the date.
2. Place the stained gel into the resealable bag.
3. Add 2 mL electrophoresis buffer and 3 drops of methylene blue electrophoresis staining solution to bag.
4. Place into a refrigerator as directed by your teacher.
5. Consult your instructor for appropriate disposal procedures.

Student Worksheet PDF

11160_Student1.pdf