DNA Extraction from Fruit

Introduction:

DNA (Deoxyribonucleic Acid) is found within the cells of all living organisms, including plants and animals. DNA molecules function as instructions to make the protein and RNA molecules that contribute to the structure and function of cells. DNA molecules are large polymers of the nucleotide bases A, G, C and T. These four bases assemble into a sequence that makes up the instructions, analogous to the way letters come together to form words, sentences, and paragraphs to convey information. The order of the nucleotides that make up the DNA molecule is important because it codes for the instructions the cell needs to make all of the other molecules that contribute to its structure and function. Extracting DNA:

The process of obtaining DNA from cells is the first step in many biochemical laboratory procedures. Researchers must be able to separate the DNA gently from the unwanted substances in the cells so the DNA is not broken up or sheared. Extracting DNA from cells may sound like a difficult task, but it is not very difficult at all. The process involves a few general steps which include mashing, filtration, precipitation, and extraction.

Mashing the fruit exposes a greater surface area from which to extract the DNA. Substances are also added that will help break-down cell membranes to release the DNA. The filtration step allows for the collection of the DNA and other cellular substances. The precipitation step allows the DNA to separate from other cellular substances. Finally, the DNA is removed from the solution. We will use salt and detergent to disrupt the cell and nuclear membranes to release the DNA. Salt also adds positive charge and neutralizes the negative charge of the DNA that is a result of the phosphate backbone. After the lipid molecules of the membranes are disrupted with the detergent we will filter the insoluble cell debris from other macromolecules that are soluble in water. Next we will add ethanol. Because DNA is insoluble in ethanol, it will precipitate out of solution.

Experimental Question: Does the amount of DNA depend on the fruit type?

Materials

10 ml DNA Extraction Buffer 1 test tube 1 – 5g piece of fruit cheesecloth 1 freezer bag rod 1 funnel 5 ml cold ethanol 1 test tube rack Procedure:

1. Place a piece of fruit in a plastic bag and seal tightly pressing out excess air.

2. Gently mash the fruit for one minute. If you are using any fruit except the ‘Fresh Strawberry”, add one mL of water with the pipette.

1 3. Add 10 ml DNA extraction buffer and seal the bag tightly again.

4. Gently massage the fruit and DNA extraction buffer for one minute (limit the foam production).

5. Filter the liquid through a cheesecloth-lined funnel into a collection tube.

6. Collect about 4 ml of filtrate. If the liquid is not moving through the filter, you can use the bottom of a test tube to press lightly and help it through.

7. Slowly add 5 ml of cold ethanol by holding the tube at an angle and carefully pouring it down the side of the test tube. You should have a clear layer on top of your fruit solution.

8. Watch as the ethanol layer will separate the DNA from the buffer. Use a rod to spool the DNA by twirling it.

9. Compare your results with other lab groups using different fruit sources and frozen or fresh fruit. (Include these results in your table.)

10. Pour the excess liquid from test tube into the waste container provided. Rinse out Ziploc bag and discard the fluid in the waste container provided then turn inside out to dry. Discard the cheesecloth (with fruit) in the garbage can. Rinse out all glassware. Clean up your area and wash your hands. Raise your hand and get teacher’s initials before sitting down.

Teacher’s Initials ______

Analysis Questions 1. The DNA extraction buffer is made up of detergent and salt. Why is there detergent in the buffer? What is the purpose of the salt found in extraction buffer? 2. What does the alcohol do? Why does the DNA rise to the top after adding alcohol? 3. Where is the DNA found in a cell? 4. What kind of results would you expect if we tested fresh vs frozen fruits? 5. Do you think there would be a difference between using vegetables instead of fruit in the experiment? Explain 6. In what real-life situation would the extraction of DNA be useful? Explain. ______FYI: If you want to do this at home, here is the buffer recipe. DNA Extraction Buffer: Enough for 100 strawberries 100 mL (3/8 cup) liquid dishwashing soap or shampoo (without conditioner) 15 grams (2 teaspoons) salt 900 mL (3 3/4 cups) water