UNIVERSITI TEKNOLOGI MARA

FAKULTI KEJURUTERAAN KIMIA

GENETICS LABORATORY (CBE 561)

NAME : NURWANI BINTI HUSSIN
GROUP : 4A
EXPERIMENT : LAB 1 : GENES IN A BOTTLE
DATE : 20 MARCH 2014
PROG/CODE : EH222
SUBMIT TO : MOHAMAD SUFIAN BIN SOAIB

No Title Allocated Marks (%) Marks
1 Abstract 5
2 Introduction 5
3 Objectives 5
4 Theory 5
5 Procedures/Methodology 10
6 Apparatus 5
7 Results 10
8 Calculation 10
9 Discussion 20
10 Conclusion 10
11 Recommendations 5
12 References 5
13 Appendices 5
TOTAL 100

Remarks:

Checked by: Rechecked by:
Date: Date:


I. ABSTRACT
This experiment has provide the great tips on extraction of nucleic acid which is both
DNA and RNA. These two nucleic acid store in the nucleolus of nucleus in a cell. Certain
substances needed in order to make the nucleic acid settle out. Focus on the onions cell and
how it works. The substance like salt, detergent and alcohol added performed different
function.
The smashed and chopped onion used has significantly gave different the result. Both
onion used to identify the correct way to get the extraction and understand the concept of DNA
extraction.





















II. INTRODUCTION
Human is made up of cell which perform as building block. In a single cell, there is an
organelle called nucleus which play its role as a headquarters and control all activities inside
that single cell. The activities are including amino acid synthesis and break down of the glucose
molecule etc. Inside nucleus, there is nucleolus where it store the genetic information of DNA.
These genetic information have a special function which are responsible for expressing
someone physical characteristic such as eyes color, height and skin color. DNA also carries
information that will be required for the cells to function which mean the cells act based on
the genetic information carried by DNA.
The DNA has a structure of double helix where it is made from two strands and spiral
shape. It consist of bases such as A, T, G and C which represent as adenine, thymine, guanine
and cytosine. Adenine pairing with thymine and guanine pairing with cytosine that hold by
hydrogen bonds and form base pairs. The number of base pairs is numerous in a DNA. Sugar
phosphate backbone hold the base tightly that actually has produced a complete DNA.

Figure 1: Illustration of DNA
The letters A, T, G and C that actually form base pairs to code up genetic information in a DNA.
But the arrangement is different for every species. Even for every human, the different in arrangement
make the criminal scene easy for the detectives to understand or imagine what was actually happen and
detect the criminal with flying colors. This is because even a letter in the arrangement is different with
another person. That is why everybody has unlikely to have same skin color or height. The base pairs
arranged to give a message that contain an information where the cells called genes can read it and
perform a certain function. The DNA located in the nucleus found to be folded and molded by protein in
a cell.
Isolation genetic materials done by scientist in order to understand certain characteristic of a
species. The study of DNA is usually for detecting bacteria and viruses in surrounding environment and
also the diagnosing disease and genetic disorders. The techniques of DNA extraction are also various in
ways.
III. OBJECTIVES
i. The objective of this experiment is to isolate genetic materials from onion.
ii. To understand the concept of protein digestion.
iii. To precipitate DNA with cold alcohol.

IV. THEORY
Isolation of genetic materials or DNA from onion can be done in various way. Certain
needed substance added during experiment for a certain reason. For example, the addition of
salt helps the DNA to precipitate when the alcohol is added. That is why people who drink
alcohol are not recommended to eat salty food which can cause high blood pressure. The fact is
the backbone of the DNA which is deoxyribose sugar linked by phosphodiester group. In the
neutral pH water, oxygen atom bonded to phosphorus negatively charged. The negativity
makes the DNA dissolved in water because high polarity. Therefore, after addition of enough
salt, the positively charge cation is equal in number with anion bonded to phosphodiester
group that make the DNA less polar to water which actually slowly becomes less soluble to
water and tend to precipitate out. In a simple sentence, the negative charge neutralize by
positive charge of salt and DNA become less polar and cannot dissolve in water.
Theoretically by comparing smashed and chopped onion, the smashed onion is more
significantly really work for the experiment. Onion surrounded by cell wall can be broken by
smashing process unlikely chopped onion. When the cell wall already broken, now it is easy for
the detergent to play its role to lyse the DNA in the nucleus. The DNA located in nucleus folded
and molded by protein and lipid of nucleus membrane. In order to expose the DNA into water,
the membrane must be broken down by using detergent. Detergent that contained the
compound SDS (sodiumdodecyl sulfate) play its role to break the lipid molecules and protein
membrane where DNA finally lyse into water. Chemically, detergent contain enzymes that
would denature the structure of protein and release all nucleic acid because later on the
detergent will form bond to the protein or lipid. Nucleic acid can be classified as DNA and RNA.
The amount of detergent use in the experiment must be in correct amount, overdose amount
of detergent make it hard to control the bubble formation and the worst part is the cell would
destroyed.



Figure 2: The detergent captured the protein and lipid of the cell membrane
Another substance that make up this experiment is alcohol. Alcohol make the DNA to
precipitate or settle out of the solution. The DNA will leave all the cellular components that are
not soluble in alcohol. DNA formed can be out from the solution using stirring rod. The cold
water is better than warm water for extraction because cold water helps the DNA intact during
the extraction process. Cold water will slow down the cellular activity and preventing the
enzyme from being destroy. Most of enzyme will undergo denaturation at the temperature of
above 40 C and the optimum temperature for enyme is around .

Figure 3: The activity of enzymes against temperature











V. PROCEDURES/METHODOLOGY
1) 100ml of mix solution containing 10ml detergent (Dynamo), 90ml distuilled water and
1,5g of table salt was prepare in a 250ml beaker.
2) The onion was cut and smashed with pestle and mortar.
3) The smashed onion mixed with 100ml mixture solution.
4) Water bath was prepared at the temperature of 55-60 .
5) The solution beaker was placed in the water bath for 10-12minutes.
6) After 10-12minutes in water bath, the beaker then was placed in the water bath for
5minutes.
7) The solution was filtered into two ways, first way solution was filtered by filter paper
which shaped into a cone and pass through into a conical flask and the second way of
filtration was manually using hand which also pass through a conical flask.
8) Isopropyl alcohol was measured as 2.5ml and was poured into a vial container and
labelled as 1 and 2.
9) The labelled 1 and 2 represented the size of onion particles, which vial 1 contained the
onion solution that was smashed by pestle and mortar and vial 2 contained onion
solution that was being cut into cube small pieces.
10) Vial 1 and 2 was placed in a centrifugal machine for 5minutes for completing the
separation step.
11) Any changes that was being observed have been identified and recorded in order to
discuss it later.













VI. APPARATUS
Apparatus Function Image
1) Pestle and mortar To smash the onion and
make it into smaller
particles

2) Vial bottle To store or keep the
sample and use it later in
the centrifugal machine

3) Detergent Break down the cell
membrane by dissolving
the lipids and proteins of
the cell by disrupting the
bonds that hold the cell
membrane together

4) Onion As the subject of this
experiment where the
DNA of this onion being
extracted

5) Centrifugal machine Use to separate materials
suspended in a liquid
medium based on the
response to the gravity.

6) Conical flask and
funnel
Multifunction in
laboratory but more easy
when there is filtration
happen where the filter
paper easily place on the
funnel so that the
solution filtrated well.

7) Filter paper Used to remove solid
particles in the mixture of
onion. It comes from
various sizes and can
purchased what size
particles the paper can
remove.
















VII. RESULT
Smashed by pestle and mortar Chopped onion
Image

Observation Seen a black thread-like DNA DNA not detected
Explaination The black thread-like recorded
was confirmed as DNA by the
lecturer. Extraction DNA of
onion successfully recorded as
the particles of the onion
already break into really small
and easy for DNA to isolate
from solution.
The DNA cannot be seen in
this case because the size of
onion was not small enough.


VIII. CALCULATION
No calculation.









IX. DISCUSSION
Based on the result tabulated, this experiment is successfully come out with the DNA. The
black thread-like was the DNA observed at the bottom vial. Onion DNA supposed to have a
color of white but perhaps during collecting the sample, the apparatus in the laboratory is not
clean enough. The solution contained onion initially added by salt which promotes the
neutralization of negatively charge ion into positive ion which cause the DNA start to
precipitate out.
Strength of detergent is various but in this experiment Dynamo brand was used in order to
promote enzyme in the detergent to break the protein and lipid of the nuclear membrane.
Enzyme in detergent called SDS works as the meat tenderizer. They have the same role in this
experiment. The exact strength scale of the detergent is not available but it is estimated
suitable for this experiment. Unlike the Am Way brand, only a small amount like 1ml is enough.
Because of insufficient of exact strength scale of detergent, perhaps the DNA come out with
black color because of the chemical inside the detergent.
emperature of water bath likely not beyond the boiling point because the enyme from
the detergent only can tolerate until the temperature of or the enyme will eentually
denatured. Filter paper also play an important role, because if the filter paper too thick, it
would trap the DNA and never drained into the conical flask. If there is no filter paper, manually
filter using hands is preferred.
The alcohol added to the onion solution which promote the DNA to precipitate out. Table
salt and alcohol is simply to have the same function but differ in mechanism and works
together to make this experiment really success. The solution once soaked in cold water in
order to reduce cellular activities and help DNA intact during extraction process.
The successful experiment is done by smashed onion, contrast with chopped onion.
Chopped onion does not show any DNA available because it is so hard for the salt or detergent
to break the cell wall. Compare to smashed onion, it already been given a strong force using
pestle and mortar to break the cell wall of the onion which then salt and detergent can carry
their role.








X. CONCLUSION
In a nut shell, it can be concluded that, the onion need to be smashed up in order to get the
precipitate of DNA sequence. Compare to chopped onion, the result will be disappointing
where the cell wall cannot be penetrated by detergent and salt. Insufficient data resulting the
DNA to have a different color which are not the true color of onions DNA.
This experiment is actually the extraction of nucleic acid which is both DNA and RNA.
Initially these two type of nucleic acid has a long and thin physical characteristic in a nucleus
but after the addition of alcohol, they clump together and precipitate out using salt right after
the nucleus membrane broken by the enzyme found in the detergent. Cold water, slow down
the activities in the cell and make everything easy to work on.

XI. RECOMMENDATION

1. During soaking the beaker in the hot water bath, the temperature must be not too hot
or overtime as referred to the procedure because the hot temperature will destroy the
enzyme.
2. The amount of detergent must be measured depends on strengthen of the detergents
itself.
3. The onion must be smashed nicely, if the onion is over smash, the cell will destroyed
because the force and smash process breaks the cell wall.
4. Filter paper used in the experiment must be suitable with the particles size of the onion
because thick filter paper will trap the cellular content and also the DNA needed for the
experiment.
5. Make sure all the apparatus used in the experiment is clean and trusted enough to
proceed the experiment because dirty apparatus will produce inaccurate result.









XII. REFERRENCE

1. Centrifugation Basics. (2002). Retrieved from Department of Biology of Bates College:
http://abacus.bates.edu/~ganderso/biology/resources/centrifugation.html
2. (1994). DNA EXTRACTION. The Gene School.
3. Holum, J. (1968). Elements of General and Biological Chemistry.
4. How to Extract DNA from Anything Living. (2012, January 24). Retrieved from Genetic Science
Learning Center: http://learn.genetics.utah.edu/content/labs/extraction/howto/
5. Kim, J. (2013, Feb Tuesday). 10 Questions You Want To Ask About Proteinase K. Retrieved from
AG SCIENTIFIC: http://info.agscientific.com/blog/bid/158711/10-Questions-you-want-to-ask-
about-PROTEINASE-K
6. Pierce Protein Biology Products. (n.d.). Retrieved from Thermo Scientific.
7. Skipor, A. (2012, June). NEWTON. Retrieved from Filter Paper:
http://www.newton.dep.anl.gov/teachers/resources/basiclab/filter.html

















XIII. APPENDICES