Materials Included In Kit

Additional Materials Required

Prelab Preparation

Place ethyl alcohol into an ice bath.

Safety Precautions

Ethyl alcohol is flammable and a dangerous fire risk; keep from flame and all sources of ignition. Ethyl alcohol is denatured and is toxic by ingestion. Provide students new clean drinking cups. Never allow students to drink or eat from an apparatus in the lab. Wear chemical splash goggles. 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. The resulting mixtures can be flushed down the drain according to Flinn Suggested Disposal Method #26b.

Lab Hints

• Enough materials are provided in this Super Value Kit for 5 classes of 30 students each (150 total students) all spooling their own DNA. The laboratory can be completed in one laboratory period.
• If your tap water has any unusual properties, use store-bought bottled water for this laboratory.
• If the DNA yield is not sufficient for spooling, try the following:
  1. Rinse your mouth more vigorously and for a longer period of time.
  2. The action of the detergent in step 5 can be enhanced by placing the test tube in a water bath at 55 °C. This enhances the action of the detergent and also helps the EDTA denature the enzymes that might digest the DNA.
  3. The alcohol used in Step 7 might be more effective if it is made ice-cold in an ice bath.
• The collection of cheek cells from inside the mouth highlights the nature of body tissue. Dead cells are continually being sloughed off on both the inside and outside of the body. Recently sloughed cells still contain their nucleus and their DNA genetic material. This DNA can be collected and, if in a forensics situation, analyzed and traced to a specific individual. Many tissues are potentially good sources for crude DNA extraction following the same basic steps utilized in this lab. Many variations of the same basic procedure have been developed and proposed. All produce varying results. The idea of extracting one’s own DNA has a certain appeal to students. If you choose to experiment with other tissues or not to do human DNA at all, other common tissues can be used following the same basic procedure.
• Banana, Kiwi fruit, onion, liver and beef thymus are all excellent tissue sources for DNA. The procedure is usually varied during steps 2–4 in this lab. The tissues are not “washed” like the cheek cells in this lab. Instead, the tissues are usually ground with a mortar and pestle or in a high speed blender. After the tissue has been macerated, it is treated with the solutions used in Steps 1 and 5. The slurry of tissue is then centrifuged or filtered to “clean out” the cell debris. The supernatant solutions are then treated with alcohol, etc. as in Step 7. The hot and cold treatments of the solutions are usually more critical with these other tissues.

Introduction

Learn how to isolate DNA from human cells. Yours!

Concepts

• DNA spooling/isolation
• Cell lysis
• Solubility

Background

Less than fifty years ago the nature of the genetic code still eluded scientists. Since then, the structure of DNA was first unraveled, it has become the most significant biological topic of the century. Understanding the structure of DNA helps to explain many life processes and leads to greater knowledge of why we are the way we are.

The process of DNA extraction is of primary importance in many fields of biotechnology. It is critical for genetic research, DNA fingerprinting, and creating recombinant organisms to produce beneficial products in the field of medicine.

The process of DNA extraction, regardless of the tissue used, usually involves the same key steps:

1. A detergent/salt solution is used to break down and emulsify the fat and proteins that make up the cell membrane. The salt causes the phosphate ends of the DNA to come closer together making it easier to precipitate the DNA out of solution.
2. Shaking or blending then breaks down the cell and nuclear membranes, releasing the DNA.
3. The solution is treated (with heat or chemically) to break down any DNase enzymes present, which can digest the long DNA strands into smaller pieces making spooling more difficult.
4. DNA is soluble in water and insoluble in ethanol. The addition of ethanol causes the DNA to precipitate and come out of solution. The DNA precipitates at the water/alcohol interface allowing it to be “spooled” onto a spooling device.

Materials

Safety Precautions

Ethyl alcohol is flammable and a dangerous fire risk; keep from flame and sources of ignition. Use only clean drinking cups for this procedure. Never ingest anything from a container that has previously been in the lab. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron.

Procedure

1. Add 1 mL (20 drops) of the 8% sodium chloride solution to the larger test tube. Set the tube aside in a test tube rack.
2. Pour 10 mL of fresh tap water or bottled water into a clean 30-mL plastic drinking cup.
3. Put the 10 mL of water in your mouth and “swish” the water around between your cheek and gums for at least 30 seconds. Spit the water back into the plastic cup. (The swishing of the water washes cells from inside your cheeks into the water.)
4. Pour several mL of the “cheek cell” water into the test tube containing the salt solution from Step 1.
5. Add 1 mL (20 drops) of the 10% SDS solution and 1 mL (20 drops) of the 0.1 M EDTA solution to the “cheek” mixture in the test tube.
6. Stopper the test tube and mix the contents of the tube by gently inverting the test tube several times. Do not shake the test tube. (The SDS breaks down the cell membrane from the cheek cells, releasing the DNA into the salt solution. The EDTA solution inactivates the DNA digesting enzymes.)
   {11171_Procedure_Figure_1}
7. Holding the test tube at a slight angle, carefully add 5 mL of 95% ethyl alcohol down the side of the test tube so that it forms a layer over the “cheek” mixture in the test tube (see Figure 1). Do not mix the water and ethyl alcohol layers.
8. Hold the test tube upright for one minute and observe what happens at the interface between the ethyl alcohol and the “cheek” solution. (The clouds of white strands are the DNA. The DNA is not soluble in ethyl alcohol, so it precipitates where the two liquids meet. Soap bubbles from the “cheek” solution may get trapped in the DNA strands.)
9. Add about 1 mL (20 drops) of 95% ethyl alcohol to the smaller, empty test tube.
10. Place a clean glass stirring rod in the test tube containing the DNA. Collect the DNA by turning the rod in one direction and thus winding the DNA strands around the rod.
11. Carefully remove the rod and DNA from the solution and transfer it to the smaller test tube containing 1 mL of 95% ethyl alcohol. Observe the DNA floating in the alcohol.
12. Consult your instructor for appropriate disposal procedures.