Joseph Chalhoub

Biol 275Lab
Inst.: Nedal Taha

DNA Isolation from Onion.

I. Introduction:

Scientists are involved with research in

finding out as much as possible about the
DNA in plants and animals. Although DNA
was discovered in the 1950’s, there still
remains a lot to be known about it, especially
how it regulates the workings of the body and
how it is used to determine the corporeal
traits that we all have.

We should always remember that DNA is just

a chemical named deoxyribonucleic acid.
Because it is a chemical, we can do reactions
with it just like we can work with any other
chemical. In this lab, we will use the chemical
properties of DNA to extract it from the cells
of onions. 
Nucleic acid isolation usually consists of
three steps : (1) disruption of cell
membranes with the cell wall in some organisms and the membrane of the subcellular nucleus to
release the nucleic acids,(2) dissociation from nucleoproteins and denaturation of protein (3)
separation of DNA from other soluble cellular components.

The purpose of this laboratory is to give you firsthand experience with DNA by isolating it from plant
tissue. You will start with whole onions and end with a relatively pure preparation of DNA, containing
literally millions of genes. Once isolated, the DNA can be stored in alcohol or dried out. It will actually be
possible for you to hold in your hands the key to an organism's development and structure.

II. Objectives

1. To become familiar with the physical properties of DNA by isolating it from living tissue
 2. To learn the purpose of each step in the isolation procedure as it relates to the physical
and biochemical characteristics of the genetic material

III. Materials:
 Fresh onions       
 Detergent
 Distilled water
 Ethanol (95%)  
 Graduated Cylinders (10mL and 100mL)
 Knife                                                                                                                 
 Beaker                                                            
 Glass stirring rod
 
Solutions:

-Lysing buffer1: 1M TRIS - 10% mL SDS

5M EDTA
5M NaCl
Distilled water

IV. Procedure:

1) Dice an onion with a knife. Half an onion should be plenty for this lab. Use a mortar and
pestle to mash the pieces of onion into a pulpy sludge.
2) Adding distilled water, homogenate in a blender for about 40seconds.
3) Transfer the blended onion into a 250mL, using a funnel.
4) Add 1mL of 10% SDS and stir.
5) Heat the solution.
6) Let the mixture cool for 5 minutes.
7) Transfer to a test tube using a 50mL pipette, and then add ethanol (ice cold) slowly
pipetting down the side of the tube.

1
This buffer solution is used in this lab for several reasons. First of all, the saltiness and acidity (pH) of the solution is
very close to that in living things; as a result, the DNA will like to dissolve into this solution. Secondly, the detergent is
added to help break down cell walls in the onion cells. Cell walls in living things are made of long polar molecules with
a “greasy” end and a charged end. Because detergent is used to break apart greasy particles in your clothes, it will
also work to tear apart the “greasy” molecules in cell walls. It will be important that these cell walls break down in
this lab, because inside the cell is where the DNA is.
 V. Observation:

A clear layer of ethanol should form on top of the

onion filtrate. (DNA is not soluble in ice cold ethanol)
therefore DNA precipitates out of the solution
becoming visible as white strings in the ethanol layer.

Why we need to chill the buffer solution:

As important as DNA molecules are to life, they are still extremely fragile, and break apart easily
when removed from cells. To slow down the rate at which the DNA breaks up, we cool down
the buffer solution to near freezing. Chemical reactions always take place slower in cold
solutions than in warm ones, because there is a lot less energy around to make the reaction take
place.

Why we need to mash the onion:

What we want to do by mashing the onion is to either break the cell walls (releasing the DNA
into the juice) or at the very least expose the cell walls so the detergent can break them down.

VI. Conclusion:

DNA molecules are highly complicated and essentials compounds in the organism, and their
method of isolation from the rest of the cell is made of indispensable yet simple stages of
breaking the cell membranes, seperationg the DNA from proteins and RNAs and finally
precipitating the DNA molecules. We can actually see the basic source of life after this
experiment.

VII. Questions:

1) Buffer: a mixture of an acid and its conjugate base, which minimizes change in the
acidity of a solution when an acid or base is added to the solution thus maintaining the
pH almost constant.

Precipitate: a solid separated from a solution in which the solid is not soluble.
 Filter: A porous material through which a liquid or gas is passed in order to separate the
fluid from suspended particulate matter.

Emulsify: to form emulsions (small globules of one liquid in another liquid with which the
first will not mix)

2) The lysing buffer is used to maintain pH and other roles:

EDTA: destabilizes the cell membrane, prevents precipitation of DNA, inhibits DNAses
and binds divalent metal ions that could form salt with anionic phosphate group of DNA.
NaCl: loosens the cell wall for increase solubility and stability of DNA, releases the
plasmid DNA and sheared cellular DNA, denatures the DNA of the cell.
SDS: anionic detergent, disrupts ionic interaction between proteins.

3)

Na+

Negatively charged DNA

DNA has a double helical structure maintained by hydrogen bonds. Sodium cations are
essential in preserving this structure because hydrogen bonds are lost in a medium with
low ionic forces.

4) Other vegetables and fruits that can be used for this lab are: Tomatoes, cherries,
bananas, pineapples, apples…

 Some fruits and vegetables contain polysaccarides which make purification of the
DNA more challenging.