Materials Included In Kit

Additional Materials Required

Prelab Preparation

1. Use scissors to cut the cheesecloth into 6-inch squares.
2. The ethyl alcohol should be ice cold (approx. 0 °C) when used. Place it in an ice bath before class.

Preparation of “Banana Juice” Solution

1. In a 1-L beaker, soak 35–50 dried banana chips in 500 mL of deionized water overnight.
2. Place the banana chip mixture into a blender.
3. Pulse the solution 4–5 times.
4. Blend on high for 20 seconds.
5. Layer four layers of cheesecloth on top of one another and place over the top of a clean, 250-mL beaker.
6. Pour part of the banana mixture through four layers of cheesecloth into the beaker.
7. Gently squeeze the cheesecloth to remove most of the banana solution leaving the solids trapped in the cheesecloth.
8. Repeat steps 5–7 as needed.
9. Seal the beaker with Parafilm M or plastic wrap.
10. Label and store in a refrigerator for up to one day.

Preparation of 1X Electrophoresis Buffer

1. Measure 20 mL of 50X buffer in a graduated cylinder.
2. Add the 50X buffer to 980 mL of deionized water in a 1000-mL Erlenmeyer flask.
3. Mix with a glass stirring rod.
4. Seal the flask with Parafilm M or plastic wrap.
5. Label and store in a refrigerator for up to one week.
6. Repeat at lease twice.

Note: Prepare enough buffer solution to allow each group to cover the gel in the chamber to a depth of 2–5 mm. Depending on the type of electrophoresis unit being used, the amount of buffer needed could be as much as 300 mL per chamber. The gel preparation requires an additional 375 mL of buffer and the DNA solution requires an additional 40 mL of buffer.

Preparation of DNA Solution

1. Measure 1 g of deoxyribonucleic acid into a 50-mL beaker.
2. Use a graduated pipet to add 5 mL of 1X electrophoresis buffer to the beaker.
3. Stir with a stirring rod until the mixture is a uniform consistency.
4. Repeat steps 2–3 seven more times until a total volume of 40 mL of buffer has been added to the DNA.
5. Seal with Parafilm M or plastic wrap.
6. Label and store in a refrigerator for up to one day.

Preparation of 1X Methylene Blue Stain

1. Measure 120 mL of the 10X methylene blue staining solution in a clean, 250-mL 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 the beaker with Parafilm M or plastic wrap.
5. Label and store in a refrigerator for up to two weeks. Note: 40 mL is enough to stain a gel in the staining tray that is provided.

Safety Precautions

Ethyl alcohol is a flammable liquid and a dangerous fire risk; keep away from flames and other sources of ignition. Sodium dodecyl sulfate solutions may be irritating to skin. Methylene blue and gel loading solution will stain skin and clothing. Wear chemical splash goggles, chemical-resistant gloves and apron. Use heat protective gloves when handling hot liquids. Electrical Hazard: Treat electrophoresis unit like any other electrical source—very carefully! Be sure all connecting wires, terminals and work surfaces are dry before using the electrophoresis unit. Do not try to open the lid of the unit while the power is on. 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.

Lab Hints

• Enough materials are provided in this Super Value Kit for 5 classes with six groups of students each. The laboratory activities can reasonably be completed in two 50-minute class periods if the gels are run at 125V. The Prelaboratory assignment should be completed before coming to lab, and the Post-Lab Questions can be completed after the lab.
• If the lab must be broken into two class periods, store the extracted banana DNA in labeled microcentrifuge tubes in a refrigerator. The banana DNA may be cut and mixed with the DNA solution and gel loading solution (Activity 2, Part C, steps 4–7) prior to storage if the ethyl alcohol has been allowed to evaporate.

Gel Preparation

• If a balance is not available, simultaneously prepare all six 0.8% gels. Measure 375 mL of electrophoresis buffer to a 500-mL Erlenmeyer flask, add the entire 3 g of agarose, and dissolve as directed.
• Use the largest well-forming comb possible, usually six wells. The banana DNA band is difficult to see.
• When casting agarose gels at least one day in advance, refrigerate the gels inside resealable plastic bags with 5 mL of electrophoresis buffer on the agarose gel.
• In order to minimize tearing, store the gels inside the casting trays with the end dams on and the well-forming comb inserted into the agarose or submerged in the electrophoresis chamber. Torn or damaged agarose gels may be melted and repoured.
• After preparing the 0.8% agarose gels for the first time, record the heating method and parameters used for future labs; for example, hot plate set at 6 for 15 minutes, stir bar on low.

Gel Electrophoresis

• 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. 20–40 minutes is a reasonable time range to expect results. The DNA included in this kit will appear as a wide band, and therefore the resolution would not be improved by running the gel at a lower voltage because the DNA has not been cut by a restriction enzyme into discrete fragments.
• The deoxyribonucleic acid (DNA) powder provided is fish sperm DNA. Since the DNA is haploid, they are less than 200 bp (base pairs) long. The band will run ahead of the bromphenol blue dye marker. The fish DNA is provided to ensure student success when running the electrophoresis because the banana DNA may not fragment sufficiently to migrate through the gel.
• Bromphenol blue migrates at the same rate as a 200–400bp DNA fragment. Xylene cyanole migrates at the same rate as a 4000bp DNA fragment. Both dyes are in the gel loading solution.

Answers to Prelab Questions

1. What is the purpose of adding detergent to the cells when isolating DNA?

   Detergent breaks down and emulsifies the fat and proteins that make up the cell and nuclear membranes.

2. What does a restriction enzyme do to DNA?

   Restriction enzymes cut the DNA at specific base pattern of DNA.

3. What is the electric charge carried by DNA fragments?

   DNA has a negative charge.

Sample Data

Describe the appearance of the DNA within the ethyl alcohol layer.

The DNA appears as white or clear threads with small bubbles. Some students may say that it has a mucus appearance.

Answers to Questions

1. List three errors that could affect the amount of DNA collected in Activity 1.

   Less DNA would be recovered if the sodium chloride, SDS, or EDTA are not mixed thoroughly with the banana juice. Less DNA would be recovered if the ethyl alcohol mixes into the sample solution. Inaccurate measurement of the banana juice would affect the amount of DNA collected.

2. Where in relation to the gel loading dyes are the bands of DNA?

   The first band of DNA will run ahead of the first dye marker. The second band of DNA will run between the two dye markers or just beyond the well depending upon the size of the banana DNA fragments. The second band of DNA may not be visible.

3. If a band of DNA is located near the well, are the DNA fragments within the band large or small? Explain.

   If a band of DNA is located near the well the DNA fragments are large. Larger DNA fragments take more time to make their way through the pores in the agarose gel.

Introduction

Biotechnology involves the manipulation of living organisms or their genetic components to produce proteins, enzymes and other products that maybe useful to humans. An example of biotechnology is the synthesis of human insulin using altered bacterial cells. Human insulin obtained in this manner causes fewer adverse reactions than the cow or pig insulin previously used for the treatment of diabetes.

Concepts

• DNA spooling/isolation
• Gel electrophoresis
• Recombinant DNA

Background

The biotechnology technique used by scientists to make human insulin or to alter the genetic makeup of any animal, plant or bacteria species is called recombinant DNA. Recombinant DNA involves four basic steps: (1) identification of the specific gene (or genes) that makes the desired protein or enzyme; (2) extraction of the gene from its chromosome; (3) splicing the gene into the DNA of a recipient organism; and (4) creation of multiple copies of the genetically modified organism (GMO). In the human insulin example described, scientists followed these steps and (1) located the two genes needed to make insulin on the top of chromosome 11 in humans; (2) used special enzymes to cut the genes out of the chromosome; (3) introduced the human genes into a bacteria’s chromosome; and (4) placed the genetically modified bacteria into culture media so it would produce human insulin. Similar recombinant DNA techniques can be used to produce vaccines, a variety of pharmaceutical drugs, and even genetically modified plants or animals.

The process of extracting chromosomal DNA from the nucleus of an organism is the same whether the source of DNA is human, plant or bacterial. First, a sample of cells is mixed with detergent to break down and emulsify the fat and proteins that make up the cell membrane. Second, salt is added to make the phosphate ends of the DNA stick together, keeping the DNA in its double helix form. Third, EDTA is added to inhibit the enzymes that normally cut DNA strands apart when a cell dies. Finally, cold alcohol is added to cause the DNA to dehydrate and precipitate out of solution. The DNA precipitates out as a colorless or white solid at the water/alcohol interface boundary and can be collected using a “spooling” device.

The DNA precipitates out as long strands. In order to separate out the desired segments of DNA, scientists add special enzymes, called restriction enzymes, to the DNA. Each restriction enzyme “looks” for a specific base pattern of DNA and then cuts the DNA at or near this place in the DNA strand. SmaI is a restriction enzyme that cuts the DNA strands between a series of three cytosines and three guanines as shown in Figure 1. SmaI cuts the DNA each time it “finds” the correct sequence of DNA. This may be just once or many times depending on the number of times the correct sequence appears on the strand of DNA. Once the DNA strand has been cut into fragments of different lengths, an analytical technique called gel electrophoresis is used to separate these fragments into different bands based on their length.

Gel electrophoresis uses electrical current and an agarose gel that acts as a “molecular sieve” to separate biological molecules based on their charge and size. DNA molecules have a negative charge. When DNA fragments are placed in an electrical field the fragments will migrate toward the positive electrode (anode) (see Figure 2). By placing the DNA fragments in a molecular sieve gel, the fragments must navigate their way through the pores in the gel in order to reach the anode. A molecular sieve contains pores, like a sponge. These pores act like a maze for the DNA fragments; the smaller fragments move faster through the pores in the gel while the large fragments “get stuck” more often and therefore do not migrate as far through the maze before the electric current is turned off and the fragments stop moving. The gel is contained in a buffer solution that prevents pH changes and thus keeps the DNA negatively charged. Before the colorless DNA fragments are loaded into the gel, they are mixed with colored dye and glycerin. Glycerin is added to the DNA fragments so that the samples will sink into the wells of the gel. The colored dyes migrate through the gel just like the DNA fragments. Typically two dyes are added— one that migrates at a rate similar to small DNA fragments, and another that migrates at a rate similar to large DNA fragments. Once the first dye migrates approximately halfway across the gel, the power to the electrophoresis apparatus is shut off, and the DNA fragments stop moving.

Since DNA itself is colorless, the gel must be stained to observe that the DNA fragments are within the gel. Methylene blue is a biological stain that binds to the DNA but not to the agarose gel. Excess stain not absorbed by the DNA fragments is rinsed away leaving a specific pattern of streaks and bands that indicate the presence of DNA fragments (see Figure 3).

Experiment Overview

The purpose of the first activity is to extract DNA from bananas. In the second activity, the extracted banana DNA is combined with a known sample of DNA and analyzed using gel electrophoresis.

Materials

Prelab Questions

1. What is the purpose of adding detergent to the cells when isolating DNA?
2. What does a restriction enzyme do to DNA?
3. What is the electric charge carried by DNA fragments?

Safety Precautions

Ethyl alcohol is a flammable liquid and a dangerous fire risk; keep away from flames and other sources of ignition. Sodium dodecyl sulfate solution may be irritating to skin. Wear chemical splash goggles and heat protective gloves when handling hot liquids. Be careful not to superheat the solution because it will NOT boil until stirred, whereupon it will boil over. Wash hands thoroughly with soap and water before leaving the laboratory. Be sure all connecting wires, terminals and work surfaces are dry before using the electrophoresis units. Electrical Hazard: Treat the electrophoresis units like any other electrical source—very carefully! Do not try to open the lid of the unit while the power is on. Methylene blue and gel loading solution will stain skin and clothing. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure

Activity 1. DNA Extraction

1. Use a clean graduated pipet to add 1 mL of the 8% sodium chloride solution to a clean 50-mL beaker.
2. Use a graduated cylinder to transfer 10 mL of the filtered banana solution into the beaker. Gently swirl the solution in the beaker.
3. Use a clean graduated pipet to add 1 mL of the 10% sodium dodecyl sulfate solution to the beaker. Gently swirl the solution in the beaker.
4. Use a clean graduated pipet to add 1 mL of the 0.1 M EDTA solution to the beaker. Gently swirl the solution in the beaker.
5. Use a clean graduated cylinder to measure 10 mL of ice cold, 95% ethyl alcohol. Holding the beaker at a 45° angle, carefully pour the ethyl alcohol down the side of the beaker so that the ethyl alcohol forms a layer over the solution in the beaker (see Figure 4).
   {11159_Procedure_Figure_4}
6. Allow the beaker to sit for three minutes while observing the interface between the ethyl alcohol and the solution. Record the observations on the Biotechnology Worksheet.
7. Place a clean inoculating loop into the interface layer and remove the DNA from the beaker. Collect the DNA by scooping it up with the loop.
8. Carefully remove the loop and DNA from the solution and transfer the DNA to a microcentrifuge tube. Repeat until most of the DNA has been recovered from the interface layer.
9. Allow the ethyl alcohol in the tube with the DNA to evaporate while casting the agarose gel (Activity 2). Note: Ethyl alcohol is less dense than the electrophoresis buffer. Ethyl alcohol would cause the DNA sample to float out of the well in the electrophoresis gel.

Activity 2. Electrophoresis

Part A. Preparation of Agarose

1. Use the balance to mass 0.48 g of agarose into a small weighing dish.
2. Use a graduated cylinder to measure and pour 60 mL of electrophoresis buffer into an Erlenmeyer flask.
3. Add the 0.48 g of agarose to the 60 mL of buffer.
4. Stopper the flask with nonabsorbent cotton or a foam plug. Use a marker or wax pencil to mark the height of the solution on the Erlenmeyer flask.
5. Dissolve the agarose by heating the flask in a microwave, hot water bath or on a hot plate as instructed by the teacher. Caution: Be careful not to superheat the solution because it will NOT boil until disturbed, whereupon it may spontaneously boil out of the flask.
   1. Microwave—30–40 seconds, stir, repeat.
   2. Hot water bath—do not boil the water.
   3. Hot plate with stir bar—do not boil or scorch the agarose solution.
6. Heat until the solution is clear and agarose appears to be fully dissolved.
7. Stir frequently with a glass stirring rod or magnetic stir bar and do not allow solution to boil for more than a few seconds. Note: When preparing agarose gels using a stirring hot plate, rotate the magnetic stir bar very slowly to diminish the number of bubbles in the agarose solution.
8. Use heat-protective gloves to remove the flask and place the flask on a heat-resistant pad.
9. Check the level of the liquid in the flask. Add deionized water, if needed, to bring the solution level up to the mark on the flask.
10. To prevent damage to the casting trays, allow the agarose to cool to 55 °C before pouring checking the temperature with a thermometer.

Part B. Preparation of Casting Tray

1. Attach the rubber dams to the ends of the casting tray or use tape to create the end walls (see Figure 5).
   {11159_Procedure_Figure_5}
2. Place the well-forming comb in the groove at one end of the casting tray.
3. Ensure the casting tray is on a level surface.
4. Slowly pour the cooled agarose solution into the assembled casting tray, being careful not to create bubbles in the gel. Use a stirring rod or pipet tip to push any bubbles to the edge of the casting tray. Only add enough agarose to submerge the tips of the well-forming comb about 2 mm—do not fill the tray to the top.
5. Immediately rinse out the Erlenmeyer flask thoroughly.
6. Allow the gel to sit undisturbed for at least 60 minutes until the gel is firm to the touch. The set gel will appear opaque and somewhat white.
7. Once the gel is thoroughly set, carefully remove the well-forming comb and the end dams by rocking it gently from side to side and then pulling it off.
8. (Optional) Slide each gel into a resealable bag, add 5 mL of buffer, and refrigerate. Note: A solidified gel can be stored in buffer in a laboratory refrigerator for up to two weeks.

Part C. Loading the Gel

1. Place the electrophoresis chamber on top of a piece of white paper on a level table or countertop. Do not move the chamber after loading the samples.
2. Place the agarose gel inside the casting tray (if necessary), insert the casting tray and gel into the electrophoresis chamber with the wells toward the cathode (–) end of the 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.
3. 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 casting tray.
4. Use the inoculating needle to cut the banana DNA in the microcentrifuge tube (step 9, Activity 1) until the DNA is no longer a solid mass.
5. Use a clean graduated pipet to add 2 drops of DNA solution to the banana DNA in microcentrifuge tube.
6. Use a clean graduated pipet to add 2 drops of gel loading solution to the microcentrifuge tube.
7. Use a clean graduated pipet to mix the electrophoresis sample by drawing the sample into the pipet by squeezing the pipet bulb and releasing it back into the microcentrifuge tube several times.
8. Use a clean, needle-tip pipet to load gel loading solution into each of the two outside wells of the gel. Dispense the sample into the well by holding the pipet tip just inside the well. The sample will sink to the bottom of the well (see Figure 6). Completely fill the two outside wells with gel loading solution. Caution: Be careful not to puncture the bottom or sides of the well. Do not draw liquid back into the pipet after filling the well. Note: The gel loading solution will act as the control sample for the electrophoresis analysis.
   {11159_Procedure_Figure_6}
9. Record the location of the gel loading solution wells in the boxes on the Biotechnology Worksheet.
10. Use a clean, needle-tip pipet to fill the remaining wells with the mixed DNA sample.
11. Record the location of the DNA sample wells in the boxes on the Biotechnology Worksheet.

Part D. Running the Gel

1. Place the lid on the electrophoresis chamber and connect 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 gas at the cathode and oxygen gas at the anode.
3. Turn off the apparatus to stop the gel when the first tracking dye is halfway across 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 casting tray and gel from the electrophoresis chamber and place on a piece of paper towel. Note: Be careful not to break or crack the gel.

Part E. Staining the Gel

1. Carefully slide the gel off the casting tray and into the staining tray. Note: Do not stain the casting tray.
2. Use a ruler to measure the locations of the gel loading dyes in each lane from the end of the gel. Sketch the location of the gel loading dyes (to scale) on the gel diagram on the Biotechnology Worksheet. Include the measurement data in the diagram.
3. Gently pour 40 mL of the 0.02% methylene blue staining solution into the staining tray. Caution: Methylene blue will stain skin and clothing.
4. Allow the gel to stain for 5 minutes.
5. Pour off the stain into the “used methylene blue” beaker. Be careful not to damage the gel. The stain may be reused.
6. To destain the gel, gently pour cold tap water into the staining tray. Note: Do not exceed 37 °C—warmer water may soften the gel.
7. Occasionally agitate the water for 2 minutes.
8. Pour off the water into a waste beaker.
9. Repeat steps 5–8 until the DNA bands are distinctly visible.
10. If the bands become too light, repeat steps 3–9.
11. Place the destained gel onto a lightbox and use a ruler to measure how far the DNA bands migrated from the wells. Sketch the locations of the bands (to scale) on the gel diagram on the Biotechnology Worksheet. Include the measurement data in the diagram.
12. Answer the Post-Lab Questions.

Student Worksheet PDF

11159_Student1.pdf