Materials Included In Kit

Additional Materials Required

Prelab Preparation

Aliquot 200 μL of loading dye solution into each of the ten microcentrifuge tubes. Practice the entire pipetting procedure so that you can demonstrate the well-loading technique with skill for your students.

Safety Precautions

Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. 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. Liquid materials from this lab can be disposed of according to Flinn Suggested Disposal Method #26b. Gels can be rinsed, dried, and stored for future use.

Teacher Tips

• Enough materials are provided in this kit for 30 students working in teams of 3 or for 10 groups of students. The activity can reasonably be completed in one 50-minute class period depending upon the number of micropipets available for use.

• Electrophoresis activities require micropipets or other pipetting devices. Since all micropipets are slightly different, no attempt is made to give directions for how to use the micropipets. Be sure you are familiar with those in your laboratory.
• Most people find that a “two-handed” approach to micropipetting works best. One hand is used to manipulate the sample while the other hand is used to steady the pipet over the well.
• In step 1 of the student procedure the Practice Gel Station can be placed into an actual electrophoresis chamber or other large bowl where the Petri dish can be submerged deeper than just a covering of water on top of the Practice Station.

Introduction

Electrophoresis test materials are usually expensive or available only in small quantity. The tests require the materials to be transferred with a micropipet and precise techniques. Practice these pipetting techniques with simulated materials before experimenting with real test materials.

Concepts

• Micropipet

• Electrophoresis gel
• Loading dye
• Micropipet technique

Background

The visualization and characterization of molecules, such as DNA, RNA and proteins, rely on a vital technique—electrophoresis. Electrophoresis is an integral step in many “hot” research areas, such as DNA fingerprinting and sequencing. Electrophoresis is a process by which charged particles (ions) move in response to an electrical force. Electrophoretic separation may be performed in free solution (moving–boundary electrophoresis) or in a solid matrix (zone electrophoresis), such as agarose or polyacrylamide gels. This exercise will focus on a practice technique commonly used in agarose gel electrophoresis.

An agarose gel is made by heating a buffered solution containing agarose. Upon cooling, the gel hardens and has the appearance of a clear gelatin. Agarose is the non-ionic component of agar and is obtained from chemically treated seaweed. It is a linear polysaccharide which, as a gel, has a relatively uniform pore size. This pore size property makes it useful for separating large molecules.

If the sample molecules are charged and subjected to an electric field, they will move through the gel at a rate that is dependent upon two basic parameters: the applied charge/mass ratio of the ions and their resistance to flow. The resistance to flow of the ions in the matrix depends on the size and shape of the ions and on the agarose concentration. The mobility of a molecule is directly proportional to its charge so that the greater the charge, the further the charged molecule migrates. The size of the molecule is inversely proportional to its mobility, therefore, small molecules travel faster than larger ones.

Since most proteins and DNA molecules are colorless, the bands of protein or DNA are not visible in an agarose gel during or after electrophoresis unless they are stained in some manner. Electrophoretic migration is followed by monitoring the movement of a tracking (loading) dye, usually bromphenol blue, through the gel. When the tracking dye has migrated a sufficient distance, electrophoresis is stopped and protein or DNA is made visible by one of several staining techniques.

In order for the migration to occur in a uniform and straight-line fashion, the materials are loaded into preformed wells in the agarose. Placing the materials into the bottom of the wells without puncturing the agarose well requires practice. The materials must be pipetted carefully into the bottom of the wells beneath the buffer solution in the electrophoresis chamber. Remember the following unit conversions when working with small samples: 1 mL = 0.001 L and 1 μL = 0.000001 L.

Materials

Prelab Questions

Watch carefully as your instructor demonstrates the well-loading technique using the specific micropipets available in your lab.

Safety Precautions

Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure

1. Add enough water to the top of the practice gel station so that the entire surface is submerged in water. The water simulates the buffer solution that is used in an actual electrophoresis chamber.

2. Using a micropipet, practice loading 10 μL of loading dye into one row of practice wells. The goal is to place 10 μL of loading dye into the bottom of each well without poking any holes into the practice gel. (The practice gel is actually more rigid than real agarose.) Once the loading dye has been drawn into the pipet tip, carefully introduce the tip to the end of the well pointing towards the middle of the well itself. The tip should break the surface of the water directly over the end of the well. Slowly expel the practice loading dye. Be careful not to push the tip into the bottom of the well. If the well is mistakenly punctured with real agarose, the sample will be lost. Practice loading dye contains sucrose, which is heavier than water and causes the sample to sink and stay in the bottom of the well.

3. Take turns using the micropipet and filling the wells.

4. Practice the technique until an entire row of wells has been successfully loaded. When all wells in a practice station are filled, rinse the practice station under slow running water, being careful not to splash any dye onto clothing or other objects.

5. The rinsed station can be used again for another practice round, if desired.

6. Consult your instructor for appropriate disposal procedures.