Materials Included In Kit

Additional Materials Required

Prelab Preparation

Make enough copies of student worksheets prior to class time. Each student group will require a Paper Gel Electrophoresis Worksheet and one of the six alternative Standard Curve Worksheets. Also locate containers to separate and hold pop beads at each student workstation.

Safety Precautions

This simulation requires no hazardous materials. Good laboratory procedures should always be followed.

Teacher Tips

• Enough materials are provided in this kit for 30 students working in pairs, or for 15 groups of students. All materials are reusable. This laboratory activity can reasonably be completed in one 50-minute class period or as homework after class.

• DNA basics should clearly be covered via other teaching strategies prior to doing this activity.
• This electrophoresis simulation is an ideal exercise prior to doing actual electrophoresis work. If actual electrophoresis materials are available, use them to augment the background information in this activity.
• Six alternative worksheets are provided so that different groups can work on different tasks or different classes can work on different samples. The alternate forms provide flexibility in their use and can be useful if the materials are used for evaluation purposes.
• If semi-log graphing is unfamiliar to students, you might enlist the help of math teachers or take time to provide assistance in this area.

Teacher Handouts

10253_Teacher1.pdf

Introduction

Gel electrophoresis is a technique that allows separation of DNA, RNA and protein segments based on size. The basic techniques are simulated in this activity.

Concepts

• R_f

• Fragment size (base pair: bp)
• Standard curve
• Electrophoresis

Background

Overview

Gel electrophoresis is a technique that involves the movement of molecules through a gelatin-like material in an electrical field. Since the distance that a molecule will move is dependent on its size, gel electrophoresis is a good technique to separate different size DNA fragments. The smaller fragments will move the farthest from the sample well. An electrophoresis apparatus has five major components: (a) electrical current; (b) the test sample (e.g., DNA); (c) the gelatin medium (agarose) that the sample moves through; (d) a liquid to conduct the electrical current (usually a buffer); and (e) a stain used to highlight the migrated samples.

The electrophoresis procedure usually involves the following key steps:

1. Test Sample Preparation—Involves isolating and purifying the samples as well as the addition of the “tracking” dye. The tracking dye provides a visual marker as the DNA samples travel down the gel. The addition of concentrated sucrose in the samples causes them to sink to the bottom of the sample wells.
2. Gel Preparation—Several types of gel materials can be used. Agarose, an uncharged polysaccharide purified from agar, is commonly used. When agarose solidifies it forms a matrix. The size of the pores in the gel matrix can be varied by using different concentrations of agarose. The higher the concentration of agarose, the smaller the pore size. A gel is cast by pouring agarose into a casting tray. A well-forming mold is inserted into the cast to create gel wells. After the agarose hardens the mold is removed and the gel is ready.
3. Gel Loading—The gel is placed into the electrophoresis chamber and a running buffer is added to the chamber, covering the entire gel. Usually a 10 μL sample is placed into the gel wells with a micropipet. The chamber is then sealed and an electrical field applied to the gel chamber.
4. Sample Tracking—The run continues until the tracking dye reaches the end of the gel. The power is turned off and the gel is removed.
5. Gel Staining—A variety of staining techniques can be used to enhance the visibility of the DNA bands left at various locations along the gel. Once stained, the gel can be refrigerated and stored. Often the gels are photographed before disposal to provide a permanent data record.

Standard Curves

The mobility of DNA fragments in an electrophoresis chamber can be standardized by running the fragments repeatedly under identical conditions (i.e., pH, voltage, time, gel type, gel concentration). Under identical conditions, identical length DNA fragments will move the same distance in a gel. Thus, the length of an unknown DNA fragment can be determined by comparing its electrophoretic mobility on an agarose gel with that of DNA marker samples of known lengths.

“DNA markers” are samples of DNA which have been degraded (digested) by enzymes known as restriction enzymes. Restriction enzymes cut DNA molecules precisely into fragments of a predictable, known size. The length of the DNA fragment is usually given in nucleotide base pairs (bp). The smaller the DNA fragment, the faster it will move down the gel during electrophoresis (see Figure 1).

Each fragment has a “relative mobility” value for identical conditions. This relative value is called the R_f value and can be expressed as:

A standard curve is usually constructed on semi-log graph paper by plotting the R_f value of each fragment versus its molecular weight (bp). This “standard curve” becomes a useful tool in determining the length of unknown samples.

Restriction Enzymes

The enzymes which break DNA molecules at internal positions are call restriction endonucleases. Enzymes that degrade DNA by digesting the molecule from the ends of the DNA strand are termed exonucleases.

Restriction enzymes have developed into one of the primary tools in molecular biology. Each restriction enzyme recognizes a specific nucleotide sequence. The enzyme “scans” the length of the DNA molecule and then digests it at a particular recognition sequence. For example, the EcoRI endonuclease has the following recognition sequence:

It breaks the DNA at the locations indicated by the dotted line and produces “ragged-ended” sequences often called “sticky ends.” Other endonucleases cut the DNA cleanly at one specific base pair.

Restriction endonucleases are named using the following convention:

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.

Materials

Safety Precautions

This activity is considered nonhazardous. Follow all standard laboratory safety guidelines.

Procedure

1. Assemble the DNA marker fragments to match the fragments on your assigned Standard Curve Worksheet. Use a single color pop bead for each fragment. Use the string tags to number and label each fragment.
2. Place your assigned marker sample fragments on the Paper Gel Electrophoresis Worksheet. The migration distance is the number of mm that the fragment will move down the gel from the well. Use a ruler to mark the position of each marker migration in the first lane of the gel. Lay each pop bead model alongside the marked positions. (Lay them horizontally across the width of the worksheet.) What trend do you see relative to size and distance?
3. In lane two of the electrophoresis gel, mark the position of the unknown DNA fragment.
4. Calculate the R_f value for each marker fragment and record it on the Standard Curve Worksheet.
5. Use the semi-log grid on the worksheet to construct a standard curve using the DNA marker data. Draw a “best fit” line for the standard curve.
6. Use the standard curve to determine the length (bp) of the unknown DNA fragment.

Student Worksheet PDF

10253_Student1.pdf