EXPERIMENT 1. DNA Extraction

Experiment 1
DNA Extraction
Name: Mhar Jo F. Mondana
09/08/2015
Section: TF 2:00-3:30 PM
Date Submitted:
I. Introduction
Deoxyribonucleic acid (DNA) is known to be the carrier of genetic
material of cells (Voet, Voet, & Pratt, 1999) hence it can replicate itself and
can synthesize RNA (ribonucleic acid). Since it is considered to be the
blueprint of all organisms genetic make-up, one of the basic techniques
known as DNA extraction is used in studying DNA. This method is important
as it helps in detecting genetic disorders, produce genetically engineered
organisms, produce DNA fingerprints, etc., (Biology Junction).
This experiment intends the use of fruits as samples for DNA extraction, to
be able to extract the DNA from the desired fruit sample and to know the
principles behind each step involving DNA extraction.
II. Materials and Methods
Preparation of the fruit sample
A guava fruit sample was sliced and weighed in the analytical balance.
After weighing, it was put in a zipper bag where entire air was squeezed out.
Then the tissue was crushed thoroughly for 5 minutes.
Extraction of DNA
The mashed bag of fruit solution was moved into the ice bath for 1
minute. Next, it was removed and was carefully mixed 5 times. Then, 2mL of
the aliquot was pipetted into a test tube.
Filtration
It was then filtered and drained for 5 minutes.
Washing and Re-suspension
Approximately 2 mL of ice-cold ethanol was carefully dropped in the
tube containing the filtered solution. Ethanol was allowed to rest at the top of
the solution for 2 minutes without agitating it.
The DNA appeared as transparent, slimy, white mucus that was spooled by
using Pasteur pipette.
III. Results and Discussion

About 27.0 grams of guava fruit slices were used as a sample. In order
to remove and purify DNA from the rest of the cell, four steps were used such
as Lysis, Precipitation, Wash, and Re-suspension. Lysis was done by mashing
or crushing the sample to break the cell wall and cell membrane especially
the plasma and the nuclear membranes. As the detergent was added to the
solution, it disrupted the cell and nuclear membranes enabling the release of
DNA.
Precipitation was made in the process of adding salt to the solution. It
is responsible for the neutralization of the negative charges present in the
DNA enabling the DNA strands to stick together causing it to fall out of
solution.
Meanwhile, cooling the fruit solution was observed since DNases are
present. These DNases or restriction enzymes destroy the DNA in order to
protect it from invasive viruses. Since detergent was used to break the cells
nuclear membrane, it is now exposed to these enzymes that will result to the
degradation of DNA. But these enzymes are sensitive to temperature that
cooling helps in slowing down the degradation process.
It was then washed by an ethanol solution that enabled the formation
of 2 separate layers since it is less soluble than water. Upon the addition of
ethanol, salts were removed and other water-soluble impurities except DNApulling up the water from the DNA molecule so it then collapsed in on itself
and was precipitated. Ice-cold ethanol was used because the colder the
ethanol, the greater amount of DNA can be precipitated.
The impurities-free DNA was re-suspended in a buffer so as to ensure
its stability. It appeared as stringy strands with transparent clumps. Clumps
were seen since each cell contains long DNA strands and in order to fit these
strands together, it should be efficiently packed so DNA twists tightly and
clumps together inside the cells.
Picture 1. Re-suspended DNA of guava fruit
Picture 2. DNA appearing as stringy strands with transparent clumps
IV. Conclusion
Thus, in order to extract the DNA from guava fruit the first step is to
lyse or open the cell. After breaking, solutions such as salt and detergent were
added that aids in the breaking down and the emulsifying of fat and proteins
making up the cell membrane. Lastly, ethanol is added preventing the DNA
from dissolving into the water and allowing DNA to fall out of the solution. Also,
it can be further narrowed down to 3 steps: Detergent, eNzymes, Alcohol.
V. Reference(s)
Voet, D., Voet, J. G., & Pratt, C. W. (1999). Fundamentals of Biochemistry. New
York: John Wiley & Sons, Inc.
Biology Junction. (n.d.). Extract DNA from Any Living Thing. Retrieved from
Biology Junction: http://www.biologyjunction.com/extracting_dna.htm
APS. (2015). Activities: Classroom Activities in Plant Biotechnology. Retrieved
from APSnet.org: http://www.apsnet.org/EDCENTER/K12/TEACHERSGUIDE/PLANTBIOTECHNOLOGY/Pages/Activity1.aspx
Kennedy, S. (2015, June 23). DNA Precipitation: Ethanol vs. Isopropanol.
Retrieved from BiteSizeBio: http://bitesizebio.com/2839/dna-precipitationethanol-vs-isopropanol/
Carboni, G. (2007, January). Fun Science Gallery. (D. Desaulniers, Editor)
Retrieved from Funsci.com: http://www.funsci.com/fun3_en/dna/dnaen.htm

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Experiment 1

III. Results and Discussion

Picture 1. Re-suspended DNA of guava fruit

Picture 2. DNA appearing as stringy strands with transparent clumps

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