Activity 4: Extracting and Isolating DNA
Objective
Students will extract and isolate DNA
from onion cells and herring sperm.
Background Information
DNA does not exist as a free molecule in a cell, but instead is associated
with proteins and RNA.
Thus, the process of extracting and isolating DNA from a cell and other
molecules is the first step for many laboratory procedures in biotechnology.
This process involves gently breaking up the cells, denaturing the proteins,
and then precipitating the DNA as a white fibrous material. The exact steps
involved in this process vary depending on the organism from which the DNA
will be extracted. The procedure used in this activity is a mod)fication
of the Marmur preparation, which is used worldwide in biotechnology laboratories.
Materials
For teacher prep:
- distilled water
- sodium chloride (0.15 M)*
- sodium citrate (0.15 M)*
- dishwashing detergent
- 2 water baths
- balance
For each group:
- 2 L beaker
- 250-ml beaker
- two small test tubes
- test tube rack
- 10-ml graduated cylinder
- 100-ml graduated cylinder
- plastic wrap
- cheese cloth or #6 paper coffee filter
- blender
- plastic wrap
- diced onions
- liquid with herring sperm DNA*
- cold isopropyl alcohol (at least 90% concentration)*
*Available through scientific or biological supply stores and catalogs.
Preparation
- Premix an extraction buffer by combining the following:
- 8.8 g sodium chloride (0.15 M)
- 32.1 g sodium citrate (0.15 M)
- 100 ml dishwashing detergent
- Dissolve with distilled water to 1 liter.
- Dice onions into small pieces.
- Set up 2 water baths, one hot and one cold. The hot water bath should
be approximately 60 degree Celsius; the cold water bath should be prepared
with ice.
- Be sure alcohol is well chilled.
Instructions
Part 1: Extracting Onion DNA
- Have students transfer 25 g of diced onion into a 250-ml beaker. They
should then add 20 ml of extraction buffer to the beaker.
- Have student place plastic wrap over the top of the beaker, place the
beaker into a 60 degrees Celsius hot-water bath, and let it sit in the
bath for 15 minutes.
- Next, have students remove the beaker from the hot water bath and put
it immediately into an ice-water bath. Students should let it sit in the
ice-water bath for 10 minutes.
- Have students place the mixture in the blender, and blend for approximately
a minute. Then have them filter the mixture by pouring it through several
layers of cheesecloth (or a #6 coffee filter) into a large beaker.
- Ask students to gently swirl the liquid in the beaker, and then have
them carefully transfer 3 ml of the liquid in the beaker into a small test
tube.
- Have students pour 5 ml of cold isopropyl alcohol down the side of
the test tube. Students should place plastic wrap tightly over the top
of the test tube, and slowly turn it upside-down and back again. They should
be able to see a white mucus-like material form. This is the DNA. If needed,
have students gently tilt the test tube several more times until they see
the DNA precipitate.
- Have students set this test tube aside and continue to Part 2.
Part 2: Extracting Herring Sperm DNA
- Supply each group with 1 ml of liquid containing herring sperm DNA.
- Have students pour the liquid into a test tube and add 5 ml of cold
isopropyl alcohol. Students should place plastic wrap tightly over the
top of the test tube, and slowly turn it upside-down and back again. They
should be able to see the white mucus-like DNA form. If needed, have students
gently tilt the test tube several more times until they see the DNA precipitate.
- Ask students to compare and contrast the herring sperm DNA and the
onion cell DNA.
Discussion Questions
- The extraction buffer you used for this experiment stabilizes the DNA
and prevents enzymes from breaking
apart the DNA. Why is this important? (Answers will vary.)
- How is the onion cell DNA similar to the herring sperm DNA? (Possible
answers include: Both types of DNA look like white mucous, both contain
the same materials, both are visible as strands to the unaided eye.) How
is it different? (Possible answers include: The nucleotide sequences are
different, one type of DNA is from plants, the other type is from animals.)
Extension
Have students draw out the threads of DNA from the onion cell and herring
sperm. To do this, supply each group with a pipette that has one end melted
to form a small hook. (You may substitute a pipe cleaner if you wish.) Have
students follow the instructions for extracting and isolating the DNA up
to Step 5. Then have students gently pour the alcohol down the sides of
the test tube. Ask students to use the pipette hook to gently tease out
a thread of DNA from the interface between the alcohol and the liquid in
the test tube. If students twirl the DNA around the hook as they draw it
out, they will find that they can isolate quite a long strand of DNA.
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