KOLEJ MATRIKULASI MELAKA

KEMENTERIAN PENDIDIKAN MALAYSIA BIOLOGY (SB015)

78300 MASJID TANAH, MELAKA SEMESTER I
2023/2024

BIOLOGY LABORATORY : MB 6

NAME : EESON WONG ZHUANG HONG

MATRIC NUMBER : MS2311101744

PRACTICUM GROUP : H1T8

LECTURER NAME : Madam Michelle

DATE (√) EXPERIMENT TITLE

1 BASIC TECHNIQUES OF MICROSCOPY
2 PLANT TISSUES
3 TRANSPORT ACROSS MEMBRANE
4 CELL DIVISION - MITOSIS
5 INHERITANCE
21.8.23 √ 6 ISOLATING DNA

REPORT CRITERIA MARKS

MANIPULATIVE SKILL (10%)
INTRODUCTION (5%)
PROCEDURES (10%)
OBSERVATIONS/RESULTS (40%)
DISCUSSIONS/QUESTIONS (20%)
CONCLUSIONS (10%)
REFERENCES (5%)
TOTAL (100%)
TITLE : Isolating DNA
OBJECTIVES : 1. To isolate DNA from plant tissue

INTRODUCTION :

DNA is a macromolecules that consists of two polynucleotide strands, where

each polynucleotide strand is made up of monomers called nucleotides. The
two strands twist around each other to form double helix. Nucleotides consists
of deoxyribose sugar, phosphate group and a 4 nitrogenous base. Both strands
are held together by hydrogen bond between complementary base pairs.
Adenine(A) pairs with Thymine(T) with two hydrogen bonds while Guanine(G)
pairs with Cytosine(C) by three hydrogen bonds. One strands ends with a free
phosphate (5’ end) while the other strands ends with a hydroxyl group (3’ end).
The bond between 3’ hydroxyl group of deoxyribose sugar and the phosphate
group is known as phosphodiester bond.

Organelles in eukaryotic cell that contain DNA including nucleus, the cell’s
“command centre” that stores the cell’s DNA. Besides, mitochondria which is
the hub of a cell’s energy production that contain a small amount of their own
DNA. In plant cells, chloroplasts contains their own genome (DNA) apart from
the nucleus .

Extracting DNA from fruit cell is difficult the animals cell due to the presence
of cell wall. Comparatively, animal cell have no cell wall to break down, thus
DNA is easier to extract from animal cell.

DNA extraction is required to get a considerable amount of copies of a

particular sequence or gene by amplification. It is needed in the industries like
inherited disease screening, microbial identification, disease diagnosis, genetic
screening, DNA fingerprinting, STR typing, and DNA or gene testing, maternal
testing, criminal investigation and plant genetic studies.

PROCEDURE : 1. The salt-detergent solution was prepared. The solution was stirred gently to
completely dissolve the salt.
2. 10ml of ice-chilled alcohol was poured into a boiling tube and placed into a
beaker containing ice cubes.
3. Watermelon given was sliced and mashed with mortar.
4. The mashed fruits was transferred into a beaker and 100ml of salt-detergent
solution was added. The mixture was incubated in the water bath at 60oC for 15
minutes.
5. The mixture was sieved with muslin cloth and the liquid was collected in a
beaker after 15 minutes.
6. Sieved liquid was filled into a boiling tube until half.
7. 10ml of ice-chilled alcohol was very carefully poured into the side of the
boiling tube at a flat angle.
8. The boiling tube was put into a rack. The extracted DNA between alcohol and
sieved liquid was observed and drew.

OBSERVATION :
DISCUSSION : Use this reference to help you to answer the given questions :
https://www.apsnet.org/edcenter/disimpactmngmnt/labexercises/
PlantBiotechnology/Pages/Activity1.aspx

1. Explain the result you obtain- colour, texture and location of DNA in
the boiling tube.
Colour : Red
Texture : Gel like layer
Location : In between alcohol and sieved liquid

2. Explain the purpose of each step below.

i. Mash/squash the fruit with mortar and pestle?

Mashing the fruit is a necessary procedure so that a result can be
obtained by the end of the extraction of DNA itself. Mashing the fruit
breaks the cell wall of the cell physically. This is so that the DNA
materials ae exposed for extraction.

i. Add mash fruit in salt detergent solution?

Salt purpose : 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.
Detergent purpose : After the cell walls have been disrupted during
mechanical mashing of the fruit, the detergent in the shampoo
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.

ii. Filter fruit mixture using muslin cloth?

To filter out the large particles that are not related.

iii. Add alcohol to sieved liquid?

Everything except the DNA will dissolve in alcohol. The alcohol pulls
water from the DNA molecule so that it then collapses in on itself and
precipitates. The DNA will become visible as mucous strands that can
be spooled with the wooden applicator stick.

5. Why do we have to use ice chilled alcohol to extract DNA?

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.

6. State two precaution you must take during the experiment.

Safety precaution : i. Be careful and slowly slice the fruit.
ii. Mash gently the fruit to prevent spill out of
 fruit mixture.

Procedural precaution : i. Stir gently when preparing salt-detergent

without producing foam.
ii. Drizzle the ice-chilled alcohol at flat
angle to ensure the ice-chilled alcohol
does not mix with the fruit extract.

CONCLUSION : Were you able to extract DNA from fruit? Are the DNA you extract from
the experiment is pure? Give reason to your answer.
Yes, I am able to extract DNA from fruit. The DNA extract from the
experiment is not pure since there are still protein that holding the
polynucleotide together which is histone protein.

Can you see the DNA under the microscope? If not, why?
The DNA cannot be seen under the light compound microscope because
the strands is at molecular level.

If the DNA is not pure, suggest one way how to purify the DNA.
Phenol chloroform extraction, normally followed by ethanol precipitation,
is the traditional method to remove protein and purify a DNA sample.

Suggest one way how to detect the presence of DNA.

UV- Vis Spectrophotometric analysis.

REFERENCE :
Extracting DNA from fruit - Reproduction, the genome and gene expression -

Edexcel - GCSE Biology (Single Science) Revision - Edexcel. (n.d.).

BBC Bitesize.

https://www.bbc.co.uk/bitesize/guides/z3mbqhv/revision/4