Extraction of DNA from Onion or Banana Cells
Introduction:
Deoxyribonucleic acid (DNA) is the genetic material of all organisms. This is the
Universality of DNA. It is the master molecule in whose structure is encoded all
information needed to create and direct the chemical machinery of life (Micklos and
Freyer, 1999).
Today, scientists can analyze DNA from minute samples of blood, hair roots and saliva.
Scientists have all the tools needed to clone an extinct animal as a woolly mammoth that
was preserved in permafrost. The debated question is: Should they?
Scientists can determine why the legendary chemist, John Dalton*, born in 1766 was
colorblind by extracting DNA from his preserved eyeballs. They can convict or exclude
rape suspects in the court of law, determine the sex and genetic disorders of unborn
fetuses, and create vaccines to prevent influenza pandemics as the one of 1918 which
killed 20 to 100 million people world-wide. ( Aids has killed 11.7 million people up to
1997.)
Pre-laboratory Questions:
1.
Where is chromosomal DNA located within the cell?
2.
Most organelles are mainly composed of 3 chemicals. Using your textbook if you
need to, list the chemical composition of the cell membrane. Draw a diagram of the
Fluid-Mosaic Model of Cell Membranes.
3.
The nucleotide is the basic unit of DNA. What are the 3 chemical groups that
make up a nucleotide? Draw a diagram of a nucleotide.
* John Dalton proposed the atomic theory of matter. That is, all matter is composed of
invisible entities called atoms. John Dalton requested that his eyes be preserved after his
death so that scientists in the 18th century can determine the cause of his
colorblindness. Scientists in the 20th century determined the cause.
Materials: (each group of 3 needs)
Each group needs:
Groups need access to:
2g salt
1ml of dish-washing liquid
1x 500, 250, 100ml beakers
1 pipette (1ml) + red filler bulb.
1 chopping knife
1 teaspoon
filter paper, Buchner funnel and water pump
cocktail sticks / tooth picks.
a 60 0C water bath
a balance (0.01g accuracy).
ice cubes
ice cold 95% ethanol
stopwatch
1/2 onion + 1/2 banana
Method:
1. For each half onion / half banana, make a solution consisting of 5ml of liquid
dishwashing detergent or shampoo and 0.75g of table salt. Put in a 250 ml beaker.
2. Add distilled water to make a final volume of 50ml. Dissolve the salt by stirring
slowly to avoid foaming.
3. Heat the 50 ml of this solution to 60 oC using a water bath.
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Chop half an onion / banana into tiny pieces on a tile.
Cover chopped onion with the 50 ml of solution from step 2.
The liquid detergent causes the cell membrane to break down and dissolves the
lipids and proteins of the cell by disrupting the bonds that hold the cell
membrane together. The detergent causes lipids and proteins to precipitate out
of the solution.
NaCl enables nucleic acids to precipitate out of an alcohol solution because it
shields the negative phosphate end of DNA, causing the DNA strands to come
closer together and coalesce.
Stir and let sit for 10-12 min. at 60 oC. During this time, press the chopped
onion mixture against the side of the beaker with the back of the spoon. (Do not
keep the mixture in the hot water bath for more than 15 minutes because the
DNA will begin to break down.)
Remove beaker. Immediately place on ice for 5 minutes. During this time, press
the chopped onion mixture against the side of the beaker with the back of the
spoon. This step slows the breakdown of DNA.
Pour mixture onto a Buchner funnel on a conical flask placed on ice. When you
filter the onion mixture, try to keep the foam from getting into the filtrate. It
sometimes filters slowly, so be patient.
Pour ~ 5 ml of filtrate into individual test tubes that are placed in ice bath. The
test tubes should be 1/3 full.
Each student needs 3 test-tubes.
1. Add cold alcohol to the test tube to create an alcohol layer on top of about 1
cm. For best results, the alcohol should be as cold as possible. The alcohol
can be added to the solution in three ways.
(a) Fill a 1ml pipette with alcohol, put it to bottom of the test tube, and release
the alcohol.
(b) Put about 1 cm of alcohol into the bottom of a test tube and add the onion
solution.
(c) Slowly pour the alcohol down the inside of the test tube with a teat pipette.
DNA is not soluble in alcohol. When alcohol is added to the mixture, all the
components of the mixture, except for DNA, stay in solution while the DNA
precipitates out into the alcohol layer.
2. Let the solution sit for 2-3 minutes without disturbing it.
It is important not to shake the test tube. You can watch the white DNA
precipitate out into the alcohol layer. There should be enough DNA to wrap on
to a tooth pick. DNA has the appearance of white mucus.
Summary and Analysis:
1.
Why do you think you chopped and blended the onion?
2.
Why is detergent/soap used to wash a greasy pan.
3. What does a greasy pan have in common with a cell membrane?
4.
Why do scientists use detergents to get clean DNA?
5.
Sodium chloride (NaCl) ionizes in solution, i.e., NaCl à Na + + Cl -. How do you
think the negatively charged phosphate groups of DNA will respond to the Na + and Cl
- ions?
6.
If you had access to a high tech laboratory, what investigations could you do with
the DNA that was extracted from the onion and what would be the benefit to an
individual?