Extraction of DNA from Onions
Deoxyribonucleic acid (DNA) contains the genetic code. It is also a good
example of a macromolecule because it is made up of four relatively similar monomers
called nucleotides. The four nucleotides are adenine (A), cytosine (C), guanine (G), and
thymine (T). When joined together, these nucleotides can make some very large
molecules. In humans, each cell contains 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 1 m of
DNA folded up in the nucleus.
This laboratory takes advantage of several physical and chemical properties of
DNA including its solubility in various solvents and physical length. Because human
beings have not been found to willingly volunteer their tissues for this type of experiment,
onions will be used as the source of DNA. As all organisms have a genetic code, 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 are 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.
Following cell disruption, as many possible of the unwanted chemicals released
by disruption must be removed, this is the second step. In this laboratory, the ability of
chloroform to denature protein so that it is no longer soluble is taken advantage of,
consequently protein can be easily removed. The chloroform has no effect on the
solubility of DNA.
The final step involves the actual removal of DNA from the 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 molecule that will stick to 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 50 g of diced (less than 3 mm3) onion into a 250 ml beaker and add
100 ml of homogenizing medium.
b
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.
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 thicknesses of cheese cloth and save
the filtered solution which contains the DNA.
2
Deproteinization
WARNING: Chloroform should not be inhaled under any circumstances. Do all
deproteinization steps in a fume hood that is working.
3
a
Pour exactly 50 ml of the filtered homogenate into a clean 250 ml flask
then add 2 ml of chloroform very gently by pouring it down the side of the
flask keeping the water and chloroform phases separate.
b
Very gently swirl the solution being careful not to totally mix the water
and chloroform. You should see a white precipitate appear at the interface
of the two solutions. This is denatured protein.
c
Carefully transfer the upper homogenate layer to a new 250 ml flask
leaving behind the chloroform and denatured protein.
d
Repeat steps a through c another four times to ensure that all protein has
been removed.
e
Transfer the homogenate to a clean 250 ml beaker being very careful to
leave all the chloroform behind.
Precipitation of DNA
a
Cool the homogenate to 10 oC on ice.
b
Very slowly add 50 ml of ice cold ethanol by pouring it down the side of
the tilted beaker. It is essential that the ethanol and homogenate form
separate layers.
c
Spool out the white stringy DNA that appears at the interface by gently
swirling a glass rod around at the ethanol/homogenate interface. Always
turn the rod in the same direction.
d
Place your DNA into the blue microcentrifuge tubes containing 100
microliters of TE buffer provided.
Exercise 2: Qualitative Analysis
By using the following procedure it is possible to test for the presence of DNA. A
positive reaction with the dipheylamine reagent (Dische) will provide one more item of
evidence that the substance you removed from the onion cells actually is DNA.
1
Start a boiling water bath using 100 ml of water in a 250 ml beaker. Be sure to
exercise caution when dealing with boiling water. If you are burned, run
cool water over the burnt area and inform your instructor immediately.
2
Number 3 test tubes 1 - 3. Into tube 1, place 1 ml of your onion DNA solution.
Into tube 2, place 1 ml of the standard DNA solution (this is a positive control).
Make a negative control with the third tube by placing 1 ml of distilled water into
it.
WARNING: The Diphenylamine reagent is very acidic. Handle it with care. If you
get any on your skin, rinse it at once with large amounts of water and inform your
lab instructor. Remove any clothing that gets diphenylamine reagent on it.
3
Add 1 ml of the diphenylamine reagent to each test tube. then place all three into
the boiling water bath for 10 min. The test tubes containing DNA should produce
a yellowish product with maximum absorbance at 595 nm. This is best viewed
against a white sheet of paper.
Materials
Equipment
Flasks, 250 ml
Ice bath
Pasture disposable long pipets
Test tubes, 10 ml
Thermometers
Water bath, 65 o C
Ice bath
Chemicals
Diphenylamine reagent: To make 100 ml
Mix 1 g of fresh diphenylamine with 100 ml of glacial acetic acid and 2.5 ml of
concentrated H2SO4. Stable for 6 months at 2 o C.
DNA Standard: 5 mg/ml of any DNA (Salmon sperm DNA is readily available).
Chloroform
Ethanol, 95 %
Homogenization buffer
To make 2 liters:
Sodium dodecyl sulfate (SDS)
100 g
NaCl
17.54 g
Sodium citrate
8.82 g
Ethyylenediamine tetraacetic acid (EDTA) 0.584 g
Add distilled water to
2L
Supplies
Cheese cloth
Onions