Learning Outcomes:
At the end of the lab, students will be able to
• Describe the structure and extraction of DNA from cells.
• Describe gel electrophoresis method to separate the DNA fragments and analyze sizes of DNA.
PART- I: Extraction of DNA from a Strawberry
Cells are the basic unit of life and make up all plants, animals and bacteria. Deoxyribonucleic acid, or DNA, is the molecule that controls everything that happens in the cell. DNA contains instructions that direct the activities of cells and, ultimately, the body.
The DNA molecule is a polymer of nucleotides. Each nucleotide is composed of a nitrogenous base, a five-carbon sugar (deoxyribose), and a phosphate group. There are four nitrogenous bases in DNA, two purines (adenine and guanine) and two pyrimidines (cytosine and thymine). A DNA molecule is composed of two strands. In each strand the nucleotides form covalent bonds between the phosphate group of one and the deoxyribose sugar of the next. From this backbone extend the bases. The bases of one strand bond to the bases of the second strand with hydrogen bonds. Adenine always bonds with thymine, and cytosine always bonds with guanine. The bonding causes the two strands to spiral around each other in a shape called a double helix.
Figure 1: Double helix model of DNA. (a) The sugar-phosphate backbones are on the outside of the double helix and purines and pyrimidines form the “rungs” of the DNA helix ladder. (b) The two DNA strands are antiparallel to each other. (c) The direction of each strand is identified by numbering the carbons (1 through 5) in each sugar molecule. The 5ʹ end is the one where carbon #5 is not bound to another nucleotide; the 3ʹ end is the one where carbon #3 is not bound to another nucleotide.
In this lab, you will isolate DNA from strawberries (kiwi or banana could be used as alternatives) using common house hold materials.
Humans have two copies of each chromosome (diploid genome). Strawberries have up to eight copies of each chromosome. The DNA extraction fluid contains detergent and salt. The detergent helps dissolve the phospholipid bilayer of the cell membrane and organelles. The salt breaks the protein chains that bind the nucleic acids. DNA is not soluble in cold ethanol and will precipitate out of solution.
Materials:
• Re-sealable plastic bag
• Strawberries (fresh or frozen)
• Dish Detergent: 2 teaspoons
• Salt: 1 teaspoon
• 1⁄2 cup of water
• Plastic cups (One cup will be used for the filtering apparatus below)
• Filtering apparatus: coffee filter and plastic cup
• Ice cold 90% or 70% rubbing alcohol
• 1 wooden Popsicle stick or plastic coffee stirrer
Procedure:
- Pull off any green leaves on the strawberries that have not been removed yet.
- Put the strawberry into the plastic bag, seal it and gently smash it for about two minutes. Completely crush the strawberry. This starts to break open the cells and release the DNA.
- In a plastic cup, make your DNA extraction liquid: mix together 2 teaspoons of detergent, 1 teaspoon of salt and 1⁄2 cup of water.
- Add 2 teaspoons of the DNA extraction liquid into the bag with the strawberry. This will further break open the cells.
- Reseal the bag and gently smash for another minute (Avoid making too many soap bubbles).
- Place the coffee filter inside the other plastic cup.
- Open the bag and pour the strawberry liquid into the filter. You can twist the filter just above the liquid and gently squeeze the remaining liquid into the cup.
- Next, pour down the side of the cup an equal amount of cold rubbing alcohol as there is strawberry liquid. Do not mix or stir. You have just isolated the DNA from the rest of the material contained in the cells of the strawberry.
- Within a few seconds, watch for the development of a white cloudy substance (DNA) in the top layer above the strawberry extract layer.
- Tilt the cup and pick up the DNA using a plastic coffee stirrer or wooden stick.
Results:
Insert your pictures here (show the DNA on the Popsicle stick/stirrer):
Post-Lab Questions – PART I:
- Match the procedure and the function:
Procedure Function
A. Filter strawberry slurry through cheesecloth _ To precipitate DNA from solution B. Mush strawberry with salty/detergent solution Break open the cells
C. Initial smashing and grinding of strawberry Separate components of the cell D. Adding of ethanol to filtered extract _ Break up proteins and dissolve cell membrane
- What are the complementary base pairs of DNA?
- How are they bonded together?
- Which part of DNA forms the backbone?
- Which group is present at the 5’ end and at the 3’ end?
PART – II: Gel Electrophoresis
Agarose gel electrophoresis is a simple method to analyze DNA preparations. We will learn about gel electrophoresis using a virtual lab. You will need a Flash player to use the virtual lab.
Click here to access the virtual lab.
Post-Lab Questions – PART II:
- Insert a picture of the gel after you complete gel electrophoresis using the virtual lab.
- What substance is used to separate DNA? What sort of a biomolecule is it?
- List the equipment that were used to separate DNA molecules.
- When an electric field is applied to an agarose gel in DNA gel electrophoresis, why do DNA fragments migrate through the gel towards the positive side?
- Why is DNA stained with ethidium bromide?
- Gel electrophoresis can be utilized along with other techniques in forensics and paternity determinations. In the experiment below, the DNA from 3 possible fathers was separated along with the child’s and mom’s DNA. Identify who might be the possible father.