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BSN - DNA Extraction Protocol

This document provides instructions for extracting DNA from kiwi or strawberry fruit. It lists the necessary materials and outlines a 7-step procedure: [1] crushing the fruit to break open cell walls, [2] heating the mixture to disrupt membranes, [3] cooling and filtering the mixture, [4] aliquoting the fruit solution, [5] adding cold ethanol to precipitate the DNA, and [6] collecting the DNA strands. The purpose of each step is also explained, such as using heat to break membranes, cooling to prevent DNA degradation, and ethanol to separate DNA from other cell components.

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Annica Lozano
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51 views3 pages

BSN - DNA Extraction Protocol

This document provides instructions for extracting DNA from kiwi or strawberry fruit. It lists the necessary materials and outlines a 7-step procedure: [1] crushing the fruit to break open cell walls, [2] heating the mixture to disrupt membranes, [3] cooling and filtering the mixture, [4] aliquoting the fruit solution, [5] adding cold ethanol to precipitate the DNA, and [6] collecting the DNA strands. The purpose of each step is also explained, such as using heat to break membranes, cooling to prevent DNA degradation, and ethanol to separate DNA from other cell components.

Uploaded by

Annica Lozano
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
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Republic of the Philippines

CAMARINES SUR POLYTECHNIC COLLEGES


Nabua, Camarines Sur

Name: Nicole Anne Pentecostes Course: BSN 1F


Instructor: Michael Nunez

DNA EXTRACTION
Materials (Note: Please improvise if materials are not available at home)

1 liter Zipper bag (one per student pair) with 20ml of extraction buffer
Skinned and freshly cut kiwi fruit (each fruit cut into 12 pieces) or one large
strawberry, allocate ~ 30 g
500 ml Beaker
Hot water plate with beaker or saucepan of water set at a constant 60 o C
Cheese cloth (cut to fit over small beaker) Tape
Cooler with ice water bath
Ice cold 95% ethanol (2 ml)
1 small test tube
wood applicator
Large transfer pipettes

Procedures

1. Add kiwi/strawberry fruit into extraction solution in the zipper bag. Close bag and
squeeze out air.
2. Crush the kiwi/strawberry thoroughly for 5 minutes. CAREFUL don’t break the bag!
3. Place the bags into the hot water bath for about 10-15 minutes, making sure the
fruit solution is fully beneath the water line. Occasionally shake the bag to evenly
distribute the heat.
4. Move the “mashed” bags of kiwi/strawberry fruit solution into the ice bath for
1 minute. Remove and carefully mix the kiwi/strawberry fruit solution again.
Repeat this procedure 5 times.
5. Tape the cheese cloth over the beakers. Filter the fruit mixture through the
cheese cloth. Combine solutions from all student groups at this point. Let the
solution drain 5 minutes.
6. Using the large transfer pipettes aliquot approximately 2 ml of the
kiwi/strawberry fruit solution into a test tube, one for each pair of
students.
7. Add approximately 2 ml of ice-cold ethanol to each tube by
dropping it slowly down the side of the test tube, allowing it
to rest on top of the kiwi/strawberry fruit mixture. Do not
agitate the solution.
8. Let the solution sit for two minutes without disturbing it. The
DNA will appear as transparent, slimy, white mucus which can
be spooled up with the wood applicator stick.

WHAT IS THE PURPOSE OF EACH STEP?

1. Why do we “crush” the kiwi/strawberry fruit?


➢ To physically breaks apart the cell walls.
2. Why do we use shampoo?
➢ To disrupts the cell and nuclear membranes of each cell to release the DNA.
It does this by dissolving lipids and proteins that hold the membranes
together.
3. What does the salt do?
➢ The salt neutralizes the negative charges on the DNA and thus enables the
DNA strands to stick together. It also causes proteins and carbohydrates to
precipitate
4. Why do we need to cool the mixture?
➢ DNases or restriction enzymes that destroy DNA are present in the cell’s
cytoplasm. They are there to protect the cell from invasion by viruses. Once
the nuclear membrane is destroyed by the soap, the DNA is now susceptible
to the DNases and will quickly be degraded. However, these enzymes are
temperature sensitive and cooling the solution slows down the process of
degradation.
5. What does the cold ethanol do?
➢ Everything except the DNA will dissolve in ethanol. The ethanol pulls water
from the DNA molecule so that it then collapses in on itself and precipitates.
The DNA will become visible as white mucous strands that can be spooled
with the wooden applicator stick.
6. Why can’t we use room temperature ethanol?
➢ The colder the ethanol is the greater the amount of DNA that is precipitated.

DISCUSSION QUESTIONS AND ANSWERS

1. To extract DNA from cells, what must you isolate it from in the case
of a plant such as strawberry?
➢ All the other parts of the cell - the cell wall, cell membrane, nuclear
membrane, mitochondria, vacuoles, endoplasmic reticulum, Golgi apparatus,
lysosomes, etc.
2. What steps did we use to extract the DNA?
➢ First, we broke apart the cell walls by physically squishing the fruit. The
chemical (detergent) process broke down the cell walls, cell membranes and
nuclear membranes. The fruit mixture was cooled to stop the DNases
released from the cytoplasm from destroying the cell’s DNA. The mixture was
filtered to separate out the large cell parts that are not needed. The DNA was
then precipitated through chemical means (the ethanol).
3. What is DNA used for when it is extracted?
➢ DNA can be used for the identification of people involved in crimes, to help
determine parentage of people and also of plants and animals, and to check
for genetic defects. For example, the DNA of these kiwi/strawberry fruits can
be compared to other samples to determine if one of them has been altered
in some fashion, such as changes that might be made to make a crop more
nutritious. DNA from one organism carrying a gene that codes for a specific
trait might also be used for transformation. The section of DNA containing
this particular gene can be inserted into a different organism so that the
altered organism now has a specific trait that it did not previously carry

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