DNA Extraction Laboratory

Introduction:
DNA is found in the vast majority of an organisms cells. This molecule functions in providing the plans for building the cells proteins. Proteins are vital to the cell, functioning as structural components as well and functional molecules of the cell. Enzymes and hormones, for example, are primarily proteins. DNA is located in three different structures within eukaryotic cells. Nuclear DNA is located in the nucleus; mitochondrial DNA is located in mitochondria, and; chloroplast DNA is located in the chloroplast of eukaryotic photosynthetic cells. By far, the greatest amount of DNA is located in the nucleus of eukaryotic cells. In this laboratory investigation, you will attempt to extract DNA from two different organisms using slightly different methods. Make sure you following the directions exactly in order to ensure success.

Method:
Although the methods used for DNA extractions varies somewhat, the goals remain the same: release nuclear DNA from the cells nucleus; release the nuclear DNA from the cell, and; to precipitate DNA out of solution so that it can be seen. Common components of a DNA extraction lab include:

Detergent
Detergent contains sodium laurel sulfate, which cleans dishes by removing fats and proteins. It acts the same way in the DNA extraction protocol, pulling apart the lipids and proteins that make up the membranes surrounding the cell and nucleus. Once these membranes are broken apart, the DNA is released from the cell.

Salt
Because each nucleotide of the DNA molecule possesses a negatively charged phosphate group, DNA is a negatively charged molecule. Therefore, DNA molecules are not attracted to each other but are repelled by the like charges they possess. Adding salt (NaCl) to a solution containing DNA neutralizes the negative charges of the DNA molecule, making the separate DNA molecules more likely to collect together and become visible. Salt is able to do this because the sodium and chloride ions separate in solution and the positive sodium ions (Na+) are attracted to the negative charges of the DNAs phosphate groups, thus neutralizing the negative charge.

Alcohol
The DNA released from the cell nucleus is dissolved in the water/detergent/specimen solution and cannot be seen. DNA precipitates out of solution in alcohol, where it can be seen. In other words, DNA is less soluble in alcohol than in water. When alcohol is added, the separate DNA molecules clump together and become visible.

Strawberry DNA Extraction

Introduction:
Strawberries cells have enormous genomes, providing a large amount of DNA for extraction. The strawberry cells are octoploid (8n) which means that each cell has eight of each type of chromosome.

Materials:
Ziploc baggie cheesecloth cold isopropyl alcohol test tube rack 1 strawberry funnel 2 large test tubes deionized water 10 ml detergent 250 ml beaker glass rod glass marking pen

Protocol:
1. Place a strawberry in a Ziploc bag and squish with your hands for two minutes. 2. Add 10 mL of detergent and 10 mL of water to the bag and squish the contents with your hands for one minute more. 3. Pour the strawberry mush through a funnel lined with cheesecloth into a beaker. Pour the strawberry solution from the baker into a test tube until the tube is about 1/8th full. 4. SLOWLY trickle the cold alcohol down the side of the tube onto the solution in the tube. The alcohol should form a layer on the surface of the solution in the tube. Add cold alcohol until the tube in half full. Do not shake or mix the tube. 5. Let the tube set. After a period of time, a white precipitate should start to appear. This is DNA! 6. Add 1 mL of alcohol to a second test tube and label the tube, Sample 1. 7. Slide a clean glass rod (you might need to clean it, rinse it with water and dry) into the test tube containing the strawberry DNA extract until it penetrates the interface between the layers. 8. Spool the DNA by rotating the rod while bringing it up and down gently. Do not stir. Large molecules of white DNA will precipitate on the sides of the rod. Reel in a good clump of fibers. These are not single DNA molecules, but huge strands of thousands of molecules twisted together. 9. With the glass rod place a sample of DNA into the 1 mL of alcohol in the sample test tube. You will use this sample to test for the presence of DNA in the third lab activity.

Human DNA Extraction

Materials:
8% NaCl solution deionized water glass rod glass marking pen cell lysis solution 2 large test tubes sterile cup graduated cylinder Parafilm, cold isopropyl alcohol

Protocol:
1. Add 1 milliliter of the 8% sodium chloride solution into a large test tube. 2. Pour approximately 10 milliliters of the bottled water into a plastic cup and swirl the water in your mouth for 30 seconds. Swirl vigorously as cell dislodged from your cheek into the water contain the DNA that will be extracted. 3. Spit the water back into the cup and pour the contents into the test tube containing the 8% NaCl solution. 4. Add 1 milliliter of the cell lysis solution to the test tube. 5. Cover the top of the test tube with a Parafilm and gently mix the contents by turning the test tube upside down and right side up. 6. Add 5 milliliters of the cold isopropyl alcohol to the test tube making sure to pour it at an angle down the side of the test tube. 7. Wait for approximately 5 minutes and watch as the DNA floats to the surface. 8. Pour 1 milliliter of alcohol into the second test tube and label the tube, Sample 2. 9. Use the glass rod to remove the DNA from the first test tube by twirling the rod in a circular motion. 10. Place the DNA into the second test tube containing the alcohol. You will use this sample to test for the presence of DNA in the following activity.

Testing for the Presence of DNA

Use the strawberry and human DNA extraction samples to test for the presence of DNA. Materials: 1 mL strawberry DNA extraction sample 1 mL human DNA extraction sample 2M ammonia solution 2 test tubes 2 pipettes 0.1M silver nitrate solution

Protocol:
1. Add 20 drops of 2M ammonia solution to the test tube containing the DNA extract. 2. Add 5 drops of 0.1 M silver nitrate solution to the test tube. 3. A white precipitate indicates the presence of purines. Purines are nitrogenous bases found in DNA molecules. Results: Sample Extract Strawberry Human Questions: 1. Were you able to see DNA extracted from strawberry and cheek cells? Is so, describe the appearance of the DNA. Positive or Negative for DNA? (+ or -)

2. Describe the role of the detergent in DNA extraction.

3. Describe the role of the salt solution in DNA extraction.

4. Describe the role of alcohol in DNA extraction.

5. What role does DNA serve in the cell?