Extraction of DNA from Onions
Deoxyribonucleic acid (DNA) contains the genetic instructions for all the proteins
in our body. These instructions are spelled our in a language called the genetic code.
DNA a good example of a macromolecule because it is a very long polymer made up of
four relatively similar monomers called nucleotides. The four nucleotides are adenine
(A), cytosine (C), guanine (G), and thymine (T). One set of human DNA made from
approximately three billion (3 x 109) nucleotides joined together. When strung together
in a linear fashion, as they are in DNA, three billion nucleotides stretch almost 1 m, so
each human cell has almost 2 m of DNA folded up in the nucleus. Why 2 m? Because our
cells have two sets of DNA, one from our mother and one from our father.
This laboratory takes advantage of several physical and chemical properties of
DNA including its solubility in various solvents and its physical length. Because most
human beings wont willingly volunteer their tissues for this type of experiment, so as a
substitute onions will be used as the source of DNA. As all organisms have genetic
information, onions have DNA in the nucleus of their cells just as humans have DNA in
theirs. The only difference is in what the DNA codes for, onions on one hand and
humans on the other.
The first step in extraction of any chemical from cells is the disruption of cells so
that the chemical is released. To do this with onion DNA, two techniques will be used in
this laboratory. First, physical disruption in a blender, and second, chemical disruption
using sodium dodecyl sulfate (SDS) which is a surfactant (detergent) that dissolves the
oily cell membrane. Other chemicals in the homogenization buffer we will use maintain
the correct pH, osmolarity, and salt concentration.
The final step involves removal of the DNA from solution. This is achieved by
taking advantage of two properties of DNA; 1) it is insoluble in alcohol, and 2) it is a
large long negatively charged molecule that will stick to positive charges on the surface
of glass. DNA that precipitates at the interface between the aqueous solution containing
DNA and an alcohol phase that has been carefully poured over the top can thus be
spooled (wound onto) a glass rod.
Method
1
Cell disruption:
a
Place 80 ml of water, 10 ml of dish washing detergent (SDS solution) and
10 g of non-iodized salt into a 250 ml beaker.
b
Add 50 g of diced (less than 3 mm3) onion Incubate in a 60 oC water bath
for exactly 15 min.
c
Rapidly cool the solution to 20 oC in an ice bath.
d
Place the cooled solution into a blender and homogenize.
2
e
Pour the solution back into the beaker (which should be cleaned first) and
let it stand on ice for 15 to 20 min.
f
Filter the homogenate through four layers of cheese cloth and save the
filtered solution which contains the DNA.
Deproteinization
a
3
Add 1 g of meat tenderizer to the strained homogenate then let stand for 5
minutes at room temperature. This step should digest most of the protein
present in the solution.
Precipitation of DNA
a
Cool the homogenate to 10 oC on ice.
b
Very slowly add 50 ml of ice cold isopropanol (rubbing alcohol) by
pouring it down the side of the tilted beaker. It is essential that the
isopropanol and homogenate form separate layers with the homogenate on
the bottom.
c
Spool out the white stringy DNA that appears at the interface by gently
swirling a glass rod around at the isopropanol/homogenate interface.
Always turn the rod in the same direction. The DNA will look like a blob
of mucus on the glass rod.
Questions you will want to address in your lab report:
What is the hypothesis you are testing in this experiment?
Is DNA a long or short molecule? How do you know this from what you did in your
experiment?
Inhalers that deliver DNAse enzymes (enzymes that digest DNA) to the lungs have been
used successfully to treat cystic fibrosis. Find out as much as you can about cystic fibrosis
and suggest a reason why DNAses might help people with cystic fibrosis?
Why do cells make DNAse enzymes?
Materials
Equipment
Beakers, 250 ml
Ice bath
Pasture disposable long pipettes
Thermometers
Water bath, 65 o C
Blenders
Chemicals
Kitchen dishwashing detergent
Isopropanol (Rubbing alcohol)
Meat tenderizer
Water
Supplies
Cheese cloth
Onions