The
objective of this lab was the extraction, visualization and comparison of DNA
extracted from a variety of fruit sources through the use of alcohol
precipitation. The class was separated into three different major groups, each
which worked with a different fruit. These groups were then sub divided again
for the analysis portion of the lab. The three different fruits involved
include kiwi, strawberry, and banana. These fruits were selected due to their
fairly high DNA content as the three fruits are hexaploid, octoploid, and
diploid in nature. This means that kiwi has six sets of chromosomes while
strawberry has eight sets of chromosomes and banana has only two sets of
chromosomes. Note that human DNA is also diploid in nature. The group which
this analysis is based upon was responsible for the analysis of the strawberry
DNA.
There
were four main procedural elements that were present in the DNA extraction
process. The first was the maceration and manipulation of the strawberry in the
presence of a DNA extraction buffer. The second was the separation of the
resultant mixture in an attempt to isolate the filtrate from the rest of the
components in the mixture. The third procedural element was the agitation of a
mixture containing a 1:1 ratio of the filtrate developed earlier and Ethanol in
an attempt to separate DNA from the filtrate. The final procedural element of
the extraction process is the separation of the DNA from the Ethanol in which
it was stored. This is done through the use of a centrifuge rotating at an
extremely high rate of speed. Once the DNA was extracted it undergoes a
staining process which results in the DNA adopting a blue colour. This stained
DNA can then be visualized and observed under a microscope at varying
magnifications.
The
first procedural element of the lab focuses on the maceration of strawberries
in the presence of an extraction buffer. The use of strawberries is based upon
the nature of their DNA, or more specifically, their chromosomes. Strawberries
possess octoploid chromosomes. The possession of eight sets of chromosomes
allows for a large amount of DNA to be contained in a very small source sample.
i.e. One small strawberry can contain enough DNA for many good sized samples. The
maceration and the use of a mortar and pestle on the strawberries is paramount
to the success of the extraction. By manipulating the strawberries the cell
membrane and nuclear membranes of the cell are destroyed. This releases the
genetic material of the strawberry so that it can be harvested later on in the
process. The extraction buffer is a safety measure that is put in place to
suspend the DNA and keep it from being dissolved by nuclease. The buffer is a
"soapy" solution that contains
EDTA. The detergent and salt in the buffer will ionize the cell membrane which
makes the DNA extraction process even easier. EDTA acts against nucleases to
keep the genetic material intact and able to be extracted. It inhibits the
occurrence of DNAsis.
Making the extraction buffer is quite easy. It is composed of 10% soap (Pantene Pro-V), 1% Sodium Chloride (Table Salt), and the rest of the solution is made up of water. These three simple ingrediants can work to inhibit the action of nucleases on the DNA.
Making the extraction buffer is quite easy. It is composed of 10% soap (Pantene Pro-V), 1% Sodium Chloride (Table Salt), and the rest of the solution is made up of water. These three simple ingrediants can work to inhibit the action of nucleases on the DNA.
Once
the strawberry mixture has been manipulated such that it is homogeneous in
appearance the next step involves the filtration of the sample. By placing the
mixture in a funnel lined with cheesecloth the solid components of the mixture
will be removed. The porous nature of the cheesecloth allows only liquid to
pass through it. The liquid that passes through the cheesecloth and is gathered
in the beaker underneath the funnel is known as the filtrate. The filtrate is a
solution that does not contain any solids of the strawberry. Contained in the
filtrate is the DNA of the strawberry, the extraction buffer, and the molecules
which make up the scent and colour of strawberries. The important factor of
this procedural element is the removal of all the solutes found in the initial
mixture. By having a completely homogeneous solution when the ethanol is
introduced the actual task of extracting the DNA will be much easier.
The
third procedural element of the DNA extraction process is the introduction of ethanol
to the filtrate. By placing ethanol in the same containment device (a test
tube) as the filtrate, DNA can be openly extracted from the solution. The ratio
of filtrate to alcohol is 1:1. Nucleic acids, specifically DNA, are water
soluble. The introduction of ethanol removes the water from around the DNA. In
the absence of water the DNA clumps and precipitates. This process will result
in the visible separation of DNA from the filtrate. The DNA will remain
suspended in the ethanol and the process can be aided through the agitation of
the test tube. This entire process is called DNA Extraction by Ethanol
Precipitation.
When
the DNA is finally separated from the test tube it will be stored in a micro
centrifuge tube along with some of the ethanol. To separate the DNA and the
ethanol the tube must be placed in a centrifuge which will spin at extremely
high speeds. This spinning motion will force the DNA, a solid precipitate, into
the bottom of the tube where it will form a pellet. This pellet is formed at
the bottom of the tube because the DNA is denser than the ethanol. The density
of the DNA when exposed to high rotational speeds forces it to the bottom of
the tube. The DNA will then be stuck there and the ethanol can be removed with
the help of a pipette. Once the ethanol is removed all that remains is the DNA
which can be stained and analyzed.
The
staining process of the extracted DNA takes place on a microscope slide. Two
drops of methylene blue were placed onto the DNA pellet which was dispensed
from the microcentrifuge tube and a cover slide was placed on top of the new
mixture. By adding a dye which is absorbed by the DNA a colour could be
associated with the presence of DNA. Once under a microscope the DNA and dye
are free to be observed under a variety of magnifications.
The
goal of this lab was to extract the DNA from a variety of fruits using ethanol
precipitation. By utilizing all of the procedural elements and the theories
behind them the lab was successful in nature. DNA was properly extracted and
analyzed through the use of dyes and visual inspection. Each fruit produced successful DNA extraction and the amount of DNA removed from each sample was relatively equal.To view the methodology
in a step by step process and see the visualized analysis as was seen in the
class please watch the video linked below the title. To recieve a downloadable PDF version of the report click on the title itself.
Just a couple of notes. My buddy Eric who has been responsible for most of the videos on this blog and the biotechnology blog is the person in the video wearing the maroon shirt. Cara, the other member of our group was seen explaining one portion of the lab process and played a role in the completion of the lab. I was the person wearing the blue sweater. We hope you enjoyed the lab and if you are feeling adventerous you could do the same experiment at home. Feel free to post a response video of yourself completing the experiment at home on Eric's youtube video.
Enjoy!
Just a couple of notes. My buddy Eric who has been responsible for most of the videos on this blog and the biotechnology blog is the person in the video wearing the maroon shirt. Cara, the other member of our group was seen explaining one portion of the lab process and played a role in the completion of the lab. I was the person wearing the blue sweater. We hope you enjoyed the lab and if you are feeling adventerous you could do the same experiment at home. Feel free to post a response video of yourself completing the experiment at home on Eric's youtube video.
Enjoy!
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