The Isolation Of DNA From Onion Cells

 

DNA (deoxyribonucleic acid) can be isolated from material
by homogenizing, deproteinizing, and precipitating it from
said material.
 
Procedure: 

The first step in DNA isolation is homogenization, which
involves breaking down and removing cell walls and
membranes. I prepared a medium consisting of 50.0 g of
sodium dodecyl sulfate (SDS) 8.77 g of sodium chloride
(NaCl), 4.41 g of sodium citrate, 0.292 g of
ethylenediamine tetraacetic acid (EDTA), and enough
distilled water to make 1 liter of solution. The
detergent-like action of SDS helps to dissolve cell
membranes and denature proteins. The NaCl/sodium citrate
buffer stabilizes the DNA by forming a Na+ shell around the
negatively charged phosphates of the DNA. The citrate
inactivates the DNAse that would otherwise break down the
DNA. 

I diced the six onions and weighed out 50.0 g into 250 ml
beakers. I then added 100 ml of the homogenizing medium to
each sample and incubated them into a 60° C water bath for
15 minutes. The heat softens onion tissue, allows the
medium to penetrate, denatures many enzymes and proteins,
and helps to prevent denaturing of DNA. I cooled each
sample in a 15° ice bath to prevent denaturing of DNA. I
blended each preparation for 45 seconds at low speed and 30
seconds at high speed. Blending breaks open cells and
releases their contents. I cooled these homogenates in an
ice bath for 20 minutes and filtered each homogenate
through quadruple-layered cheesecloth. These preparations
were stored at room temperature until further
experimentation continued.
 
The second step in DNA isolation is deproteinization. This
further purifies the DNA by removing proteins left from the
homogenization step. First, I poured 50 ml of each
homogenate into 50 ml flasks and added 2 ml of chloroform
to each homogenate. After swirling the mixture, the
chloroform forms a layer (phase) below the homogenate and
denatured proteins collect in-between the other two phases.
I poured the homogenate into another 50 ml flask and
collected waste chloroform and proteins in a glass jar. I
repeated this chloroform cycle for each homogenate 4 more
times, for a total of 30 chloroform cycles. For the last
cycle of each homogenate, I was careful to leave behind all
of the chloroform layer, because the chloroform would
contaminate the homogenate. I stored the purified
homogenate at room temperature. 

The last step in DNA isolation is the actual precipitation
of the DNA. I cooled each deproteinized homogenates in a
15° ice bath and then added 80 ml of -10° ethanol to each
homogenate. A layer of DNA formed between the ethanol and
homogenate, and I wound up the DNA onto glass rods, placed
the rods in test tubes filled with ethanol, and stored them
at 8° in a refrigerator.
 
I had planned not only to prove that I could extract DNA
from different species of onions, but also to prove that
DNA from each species of onion would be different. I hoped
to make a "bar-code" of each type of onion species by
separating its deoxyribonucleic acid into a pattern of
bands through a technique called gel electrophoresis.
 
The main steps in electrophoresis are: 1) resuspend the
DNA, 2) add the buffer and enzymes, 3) make the gel, 4)
load and run the gel, 5) stain, view, and photograph the
gel.
 
To resuspend the DNA, I tore off a small piece of DNA using
dissecting forceps and added approximately 40 m l of
distilled water. I extracted about 10 m l of my DNA
solution and added approximately 1 m l of two restriction
enzymes, Bam Hl and Eco Rl, along with 1 m l of
corresponding reaction buffer (each substance should be at
-20° ). I allowed approximately 30 minutes for my
restriction enzyme to work, and then added 1 m l of Final
Carolina Blu, an alternative to the more famous carcinogen
ethidium bromide. These samples were now ready to be placed
into the finished gel. 

My gel was made according to directions received within the
package, by adding 0.24 gm of Agarose along with 0.6 ml of
buffer and 29.4 ml of distilled water for each cube. I used
two cubes in my experiment, bringing my total up to 0.48 gm
of Agarose along with 1.2 ml of buffer and 58.8 ml of
distilled water. This mixture was heated in the microwave
until clear, and then allowed to cool to 55° C while
swirling the solution. I carefully removed my gel, and slid
each cube into the proper electrophoresis chamber. I filled
each chamber with enough buffer to cover the indents of
holes created by the comb placed within the gel. At this
point in time my chamber was hooked up and prepared to run
a cycle. 

Each sample was loaded into every other hole (since there
were 12 holes and only 6 samples). The chamber was placed
within an electric field, and each sample was allowed to
run for 1 hours time. At this point in time there was no
banding showing, and I became worried as to the final
results. The test was performed to the time of 3 hours, and
each gel was stained and washed (destained). I found at
this time that my gel showed no banding whatsoever, and
this is also the time when I noticed my buffer and
separated and moved in particles towards the positive (+)
end. There are several explanations as to why this part of
experiment did not work, as described in the next
paragraph. 

One of the main reasons I feel the second half of my
experiment was unsuccessful was the improper tools used.
Because I was unable to locate a micropipette at the time,
transfer pippetes were used. These instruments are not
precise, and a significant amount of my sample was wasted
in the process. Because of this I feel an insufficient
amount of DNA was transferred into each well, therefore the
tiny strands that did run the process did not show up. 

Another possibility is that the buffer was improper, as
shown by the fact that it separated and moved towards the
positive end. Because electrophoresis requires a large
amount of buffer, capsules were used that dissolved in
water (a cheap alternative). After the experiment was
completed, I noticed that these capsules had expired. This
is another factor that may have contributed to the failed
experiment. However, I accidentally proved that these
Hydrion buffer molecules have a negative charge, which is
why they traveled to the positive electrode.
 
I attempted to reconstruct my experiment using better tools
and new materials. At this point in my experiment I used
much better equipment (a micropipette) and a buffer that
came highly recommended by Edvotec, Tris-acetate-EDTA (20
mM tris, 6 mM sodium acetate, 1 mM of disodium
ethylenediamine tetraacetic acid). This 50x buffer was
diluted by adding 6 ml to 294 ml of distilled water, making
my total buffer count 300 ml. I ran my gel for the time of
18 hours (I had originally assumed I was dealing with a 25v
machine, when in reality its output was about 8 volts). 

Both gels were stained for 30 min. and destained for 45
min. At this point in time, 2 sets of bands showed on the
yellow onion gel, and 3 bands were showing on the red onion
gel. The first set of bands showing on the yellow onion gel
were very dark, and located 3.15 cm from the well from
which they originated. The second, much fainter set of bars
were located 4.45 cm from the well from which they
originated. In each yellow onion tested, the pattern
remained the same. The red onion samples produced a
three-bar pattern. The first set of bars were once again
the darkest (about 2.80 cm from well). The second bar set
was lighter, and located about 3.95 cm from the well. The
third and final set was the lightest, approximately 5.30 cm
from the well. By repeating this experiment, I was able to
prove that the DNA pattern for each onion species was
different, and members within that species share that
pattern. 

Conclusion:
 
DNA can be isolated from its surrounding matter through a
process involving homogenization, deproteinization, and the
eventual separation of DNA. "Hydrion" molecules have a
negative charge, as accidentally demonstrated by their
attraction to the positive electrode in an electrical
field. Each species of onion plant has its own DNA pattern,
and members within each species share a pattern.