Materials Included In Kit

Additional Materials Required

Prelab Preparation

Preparation of 1X TAE Electrophoresis Buffer

1. Measure 20 mL of 50X TAE buffer in a graduated cylinder.
2. Add the 50X buffer to 980 mL of distilled water in a 1000-mL Erlenmeyer flask.
3. Mix well with a glass stirring rod or on a magnetic stirrer.
4. Seal with Parafilm M or plastic wrap.
5. Label and store in a refrigerator.
6. Repeat, if necessary.
   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 unit 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. Prepare fresh buffer weekly.

Preparation of 1X Methylene Blue Electrophoresis Stain

1. Measure 40 mL of the 10X methylene blue staining solution in a graduated cylinder.
2. Add the staining solution to 360 mL of warm distilled water in a 500-mL Erlenmeyer flask.
3. Mix well with a glass stirring rod or on a magnetic stirrer.
4. Seal with Parafilm^® M or plastic wrap.
5. Label and store in a refrigerator.

   Note: Each student group will need about 40 mL of 1X staining solution to stain a gel in the staining tray (weighing dish) provided.

Safety Precautions

Wear chemical splash goggles or safety glasses whenever working with chemicals, heat or glassware, such as the staining solution or hot agarose. Use heat-resistant gloves or “hot hands” heat protector to transfer hot liquids. Electrical Hazard: Treat electrophoresis chambers like any other electrical source—very carefully! Be sure that all connecting wires, terminals and work surfaces are dry before using the electrophoresis unit. Do not open the lid of the unit while the power is on. Exercise caution in handling methylene blue and other stains or dyes—they will stain skin and clothing. Remind students to wash their hands thoroughly with soap and water before leaving the laboratory. Please review 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. TAE buffer, diluted methylene blue and excess DNA may be rinsed of down the drain with 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 kit for 6 groups of students. Both parts of this laboratory activity can reasonably be completed in two 50-minute class periods. The prelaboratory assignment may be completed before coming to lab, and the data compilation and questions may be completed the day after the lab.
• This lab will take two 50-minute class periods. Part A can be completed on day 1 and Part B may be completed on day 2.
• Once the gel has hardened, it may be stored in the refrigerator for up to two weeks in 1X TAE electrophoresis buffer.
• The gel must sit at least 20 minutes undisturbed so it will properly solidify; however, one hour is optimal.
• Part B may take more than one lab period depending on how long the gel takes to run. The gel also may require staining overnight if the bands are not visible after the first stain.
• Three lab groups may share one agarose gel if electrophoresis equipment is limited. It is a good idea to have each group make a gel, whether they will be used or not, to account for other groups’ errors.
• Sometimes the bands are not visible after the initial stain. If so, repeat the staining procedure leaving the 1X methylene blue on the gel overnight.

Teacher Tips

• Extend the lesson by incorporating a historical or technology research project prior to the laboratory.
• Flinn Scientific offers a wide variety of biotechnology and electrophoresis kits, including DNA Forensics (FB1798), DNA Paternity Testing (FB1799), and The Genetics of Cancer (FB1800).

Further Extensions

• Extend the lesson by incorporating a historical or technology research project prior to the laboratory.
• Flinn Scientific offers a wide variety of biotechnology and electrophoresis kits, including DNA Forensics (Catalog No. FB1798), DNA Paternity Testing (Catalog No. FB1799) and The Genetics of Cancer (Catalog No. FB1800).

Answers to Prelab Questions

1. Why are the sample wells of the agarose gel positioned in the gel box so that they are closest to the cathode (negative charge)? DNA is negatively charged. When the power supply is turned on, the negatively charged DNA will migrate or diffuse through the gel toward the anode (positive charge).
2. The restriction enzyme EcoRI recognizes the 5′-GAATTC-3′ sequence. How many restriction fragments will be produced when the DNA molecule shown is reacted with EcoRI?
   {10971_PreLabAnswers_Figure_7}
   EcoRI will make two cuts producing three fragments as shown.

Sample Data

Observations
Students should draw the bands they observe in their gel. The EcoRI pattern should be different from HindIII.

Sketch the DNA fragments patterns produced by both DNA restriction enzyme digests (EcoRI and HindIII).

The smallest two fragments produced by HindIII, 564 bp and 125 bp, will run off the gel due to their small size.

Answers to Questions

1. 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. 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.

2. When analyzing the DNA banding pattern, where on the gel would you expect to find the largest fragments produced by the restriction enzyme?

   Near the top of the gel closest to the well. The larger fragments move the shortest distance.

3. Compare and contrast the DNA fragment patterns obtained with EcoRI and HindIII?

   Both samples of DNA were cut in several locations. However, the size of the bands were different which was witnessed by the distance they travelled on the gel.

4. A DNA-restriction enzyme digest is analyzed by electrophoresis—it produces a large number of bands very close together. What does this indicate about the number of restriction sites in the original DNA sample?

   It would indicate that there are several recognition sequences in the DNA sample. Therefore the restriction enzyme cuts at all the restriction sites and produces several pieces of DNA.

5. Following are the restriction maps of Lambda DNA cut with EcoR1 and HindIII. The vertical lines represent restriction site locations. Lambda DNA is 48,502 base-pairs long. All numbers are expressed in units of base pairs

   {10971_Answers_Figure_6}
1. Based on the restriction site locations, determine the number and length of fragments that should be produced in each lambda DNA sample digest.

   EcoRI — 21,226 bp, 4,878 bp, 5,673 bp, 7,421 bp, 5,804 bp, 3,530 bp

   HindIII — 23,130 bp, 2,027 bp, 2,322 bp, 9,416 bp, 564 bp, 125 bp, 6,557 bp, 4361 bp

2. Label each band in the sketch of your gel with the correct location and size. Tip: If not all bands are visible on the gel add them in the approximate location on the graph where they should have appeared.

   See gel in the observation section above.

Introduction

Cloning genes and genetic engineering would not be possible without the use of restriction enzymes. Restriction enzymes recognize specific base-pair sequences and cut the DNA at those sites. Using electrophoresis to separate DNA fragments obtained in this manner is helpful to study a particular gene of interest.

Concepts

• Gel electrophoresis
• Genes
• Restriction fragment length polymorphism (RFLP)
• Restriction enzymes

Background

Restriction enzymes cut double- or single-stranded DNA at specific recognition sequences known as restriction sites. Originally discovered by researchers studying bacteria, base-pair restriction enzymes serve to protect bacteria against foreign DNA from other organisms, such as phages or other bacterial cells. Inside a bacterial host, restriction enzymes work by cutting foreign DNA by a process known as restriction.

The majority of restriction enzymes recognize and cut DNA at symmetrical sites in which the same 5′ → 3′ sequence of 4–8 nucleotides is found on both antiparallel strands of DNA. (The sequences occur in the opposite direction on the double-stranded DNA.) Restriction enzymes hydrolyze or break phosphodiester linkages in both strands of double-stranded DNA. The target sequence of a particular restriction enzyme often occurs several times within a single long DNA molecule. Therefore, the restriction enzyme will typically make several cuts throughout a DNA molecule. Since the average human DNA sequence is over 3.2 billion base-pairs long (approximately 30,000 genes), as many as 750,000 DNA fragments may be obtained after treatment with a restriction enzyme. The process of using restriction enzymes to cut the long strands of DNA is called restriction fragment length polymorphism (RFLP). The pieces of DNA that result from restriction enzyme cuts are known as restriction fragments. The restriction enzyme HbaI, for example, recognizes the sequence shown in Figure 1 and cuts the DNA between the bases marked by arrows.

The RFLP fragments are separated by a process known as gel electrophoresis. Electrophoresis is an analytical method using an electric field for the separation, identification, and analysis of charged biological molecules, including DNA, RNA and proteins. A gel is made from agarose, which is a refined form of agar. The agarose gel is positioned between two electrodes in an electrophoresis chamber, with the wells adjacent to the cathode (negative electrode). The gel acts like a molecular sieve, creating a maze which the fragments must move through on their way toward the anode (positive electrode). Smaller fragments move faster through the holes or pores in the gel while larger fragments move more slowly because of their size. When a voltage is applied to the electrodes, the negatively charged DNA fragments move toward the anode. The electrophoresis chamber is filled with a buffer solution, bathing the gel in a liquid that shields the system from changes in pH.

The DNA fragments are white to colorless and are invisible in the gel. Molecular biologists add colored tracking dyes so they can observe the rate at which DNA fragments move through the gel. Typically, two dyes are added—one that migrates at a rate similar to the smallest DNA fragments, and another that migrates at a rate similar to the largest DNA fragments. After the first dye has migrated to within 1 cm of the end of the gel, the power is shut off to the electrophoresis chamber. All DNA fragments stop migrating because the electromotive force stops. The agarose gel is then stained using methylene blue solution, which binds to DNA and allows the the bands to be seen.

Experiment Overview

The purpose of this laboratory activity is to observe and identify the DNA fragmentation patterns obtained with two different restriction enzymes. The first DNA sample has been digested with the restriction enzyme EcoRI. The second DNA sample digest was obtained using with the restriction enzyme HindIII. The samples will be analyzed by electrophoresis in an agarose gel in order to observe the different DNA fragments produced by each enzyme.

Materials

Prelab Questions

1. Why are the sample wells of the agarose gel positioned in the gel box so that they are closest to the cathode (negative charge)?
2. The restriction enzyme EcoRI recognizes the 5′-GAATTC-3′ sequence. How many restriction fragments will be produced when the DNA molecule shown below is reacted with EcoRI?

   5′- AATTCAGGAATTCTATTTACGCGATCGAATTCAA- 3′
   3′- TTAAGTCCTTAAGATAAATGCGCTAGCTTAAGTT- 5′

Safety Precautions

Wear chemical splash goggles or safety glasses whenever working with chemicals, heat or glassware, such as the staining solution or hot agarose. Use heat-resistant gloves when handling or pouring hot agarose solution. Be careful not to superheat the agarose solution because the hot solution will NOT boil until it is stirred, whereupon it will boil over. Electrical Hazard: Treat electrophoresis chambers like any other electrical source—very carefully! Be sure that all connecting wires, terminals and work surfaces are dry before using the electrophoresis unit. Do not open the lid of the unit while the power is on. Exercise caution in handling methylene blue and other stains or dyes—they will stain skin and clothing. Wash hands thoroughly with soap and water before leaving the laboratory. Please follow all laboratory safety guidelines.

Procedure

Part A. Day 1

Preparing the 0.8% Agarose Gel

1. Place a sheet of weighing paper on a 0.01-g precision balance and press the tare button.
2. Using a spatula transfer 0.48 g of agarose to the balance.
3. Add the 0.48 g of agarose to a 125-mL Erlenmeyer flask.
4. Using a 100-mL graduated cylinder, measure and add 60 mL of 1X TAE electrophoresis buffer to the flask.
5. Add a stir bar and place the flask on the magnetic stirrer hot plate.
6. Adjust the heat setting of the magnetic stirrer hot plate to medium and set the stir function on low.
7. Allow the solution to heat until all of the solid agarose has dissolved and the solution is transparent. Note: If the agarose is not completely dissolved the gel will not solidify.
8. Once the solution is transparent allow it to cool to 55 °C before pouring the agarose solution into the casting tray.

Prepare the Casting Tray

9. Attach the rubber dams to the ends of the casting tray or use tape to create end walls (see Figure 2).
   {10971_Procedure_Figure_2}
10. Place the well-forming comb into the groove at one end of the casting tray.

Casting the Gel

11. Ensure the casting tray is on a level surface.
12. Slowly pour the warm 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. Add only enough agarose to cover the tips of the well-forming comb to a depth of 2 mm—do not fill the casting tray to the top. Some casting trays may require less than 60 mL of agarose solution.
13. Immediately rinse the Erlenmeyer flask and stir bar.
14. Allow the gel to sit undisturbed for at least 20 minutes until the gel is firm to the touch. The set gel will appear opaque and somewhat white.
15. Once the gel is thoroughly set, carefully remove the well-forming comb by rocking it gently from side to side and then pulling it upward. Slide a scalpel or other thin instrument between the end dams and the gel to release the gel from the end dams. Remove the end dams.
16. Slide the gel into a resealable bag, add 50 mL of 1X TAE electrophoresis buffer, and refrigerate.

Part B. Day 2

Loading the Gel

1. Assemble the electrophoresis apparatus according to the manufacturer’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. Note: Do not move the unit after the DNA samples have been loaded (step 10).
3. Gently slide a gel from a resealable bag into the casting tray.
4. Carefully position the gel and tray into the electrophoresis chamber with the wells toward the cathode (–) end of the unit. 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 chamber to submerge the entire gel surface to a depth of 2–5 mm. If the gel begins to float away, reposition the gel on the casting 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 (–) electrode and wells located to the left, load the contents of the first DNA sample into the well closest to you. Consequently, when the gel is turned so that the wells are on the top, “1” will be in the upper left corner.
7. Obtain the microcentrifuge tube containing lambda DNA cut with the EcoRI restriction enzyme.
8. Lightly tap the bottom of the microcentrifuge tube on the tabletop to mix the contents.
9. Withdraw 10 μL of DNA from the 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 (see Figure 3). Be careful not to draw the sample further up into the pipet.
   {10971_Procedure_Figure_3}
10. Dispense the sample into the first well on the left by holding the needle tip of the pipet 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 the liquid back into the pipet after dispensing the sample (see Figure 4).
    {10971_Procedure_Figure_4}
11. Repeat steps 7–10 using the lambda DNA sample that was cut with the HindIII restriction enzyme. Use a clean needle-tip pipet and load sample into well 2.

Running the Gel

12. Place the lid on the electrophoresis chamber and connect the unit to the power supply according to the manufacturer’s instructions. Note: Bubbles should be observed next to 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 the oxygen at the anode.
13. Set the power supply at 125 V. Typically, the power supply is turned off when the first tracking dye is within 1–2 cm of the positive end of the gel. This should take around 30 minutes.
14. Turn off the power to the unit, then remove the cover and carefully remove the gel tray from the chamber.

Staining the Gel

15. Slide the gel out of the casting tray and into a large weighing dish which will serve as the staining tray. Note: Do not stain the casting tray.
16. Gently pour 40 mL of 1X methylene blue staining solution into the staining tray.
17. Allow the gel to stain for 5–10 minutes.
18. Pour off the stain into a glass beaker or storage bottle. The stain may be reused. Exercise caution not to damage the gel.
19. To destain the gel, gently pour cool tap water into the weighing dish. Note: Do not exceed 37 °C; warmer water may soften the gel.
20. Occasionally agitate the water for 10 minutes.
21. Pour off the stain water into a waste beaker.
22. Repeat steps 16–21 as needed until blue DNA fragment bands are distinctly visible in the gel.
23. If the bands are still not visible after the destaining process consult your instructor for directions for an overnight staining procedure.
24. Place the gel on a light box, if available. Observe the DNA band pattern for both lanes of DNA and record observations on the Roles of Restriction Enzymes Worksheet.
25. Consult your instructor for appropriate disposal procedures.

Student Worksheet PDF

10971_Student1.pdf