Universiti Teknologi MARA Cawangan Perak

Kampus Tapah

Faculty of Applied Sciences

Diploma in Science

BIO301

Video-based Practical Experiments

Practical 1: Techniques in Microbiology
Practical 2: Techniques in DNA Technology

Lecturer:
Madam Wan Nurul Hidayah Wan Anuar

Group:
A4AS1205_3

No. Name of Students Student No.

1. Aina Nabila Binti Noor Izhan 2019427262
2. Nur Ain Sofiya Binti Zulfaisal 2019213634
3. Nur Faqihah Hasanah Binti Meftahuddin 2019620756
4. Wan Mohamad Syazwi Bin Mohamad 2019263696
5. Ahmad Izzuddin Bin Zahid 2019448004
6. Noorfahmy Bin Ahmad Suhaimi 2019648902
Table of Contents
PRACTICAL 1: TECHNIQUE IN MICROBIOLOGY........................................................................................ 3
INTRODUCTION............................................................................................................................... 3
OBJECTIVES.......................................................................................................................................... 5
PROBLEM STATEMENT........................................................................................................................ 5
HYPOTHESIS......................................................................................................................................... 5
MATERIAL AND APPARATUS................................................................................................................6
PROCEDURE......................................................................................................................................... 7
RESULT................................................................................................................................................. 8
DISCUSSION....................................................................................................................................... 11
CONCLUSION......................................................................................................................................15
REFERENCES.......................................................................................................................................16
EXPERIMENT 2: TECHNIQUE IN DNA TECHNOLOGY..............................................................................18
INTRODUCTION..................................................................................................................................18
OBJECTIVES........................................................................................................................................ 19
PROBLEM STATEMENT...................................................................................................................... 19
HYPOTHESES...................................................................................................................................... 19
MATERIALS & APPARATUS.................................................................................................................20
PROCEDURE....................................................................................................................................... 22
RESULTS............................................................................................................................................. 26
DISCUSSION....................................................................................................................................... 27
CONCLUSION......................................................................................................................................30
REFERENCES.......................................................................................................................................31
PRACTICAL 1: TECHNIQUE IN MICROBIOLOGY

INTRODUCTION

Aseptic technique is a method for reducing microbial contamination that incorporates target-
specific practises and procedures carried out under carefully controlled settings. Conducting
research in the subject of microbiology requires this laboratory competence. ( Shafiquzzaman,
S. , 2017). The objectives of this experiment are to determine several techniques in
microbiology and to examine macroscopic and microscopic characteristics of Lactobacillus.
Furthermore, there are a few principles of streaking technique. Firstly, on solid media, an
original inoculum containing a combination of bacteria is dispersed into four quadrants. A
culture medium is a liquid or gelatinous substance that contains essential nutrients, to
cultivate target microorganisms or tissues, for further purposes. A culture medium must be
sterilised before use so that no unwanted microorganisms grow, which may contaminate the
growing sample. Streaking is a technique used to isolate a pure strain from a single species of
microorganism, often bacteria. Samples can then be taken from the resulting colonies and a
microbiological culture can be grown on a new plate so that the organism can be identified,
studied or tested. To reduce the number of bacteria in each subsequent quadrant is the goal of
the technique. From streaking method, the colonies are masses of offspring from an
individual cell. Individual cells are split into discrete colonies, which replicate to form
independent colonies in the later quadrants. Based on this experiment, is the techniques in
microbiology gives an impact to examine the characteristics of Lactobacillus? The hypothesis
of first experiment is if we learn the techniques in microbiology, then we can examine the
characteristics of Lactobacillus

Next, in microbiology, Gram staining is the most common, important, and widely utilised
differential staining technique. (Sagar A. , 2018) This test separates bacteria into Gram
Positive and Gram Negative bacteria, which aids in microbial categorization and distinction.
The function of reagent used in the experiment which are crystal violet is the Primary stain,
and is used to color both Gram-positive and Gram-negative bacteria purple. Next, iodine is
used as a dye-fixator (mordant) in Gram-positive and Gram-negative bacteria, removing the
purple stain. Other reagents are a decolorizer made of acetone and alcohol and Safranin, the
counterstain staining the Gram-negative bacteria, pink. Despite the addition of the pink
safranin, the Gram-positive bacteria are already purple due to the deeper Crystal Violet. From
the explanation given, we can know the hypothesis of this experiment is if the bacteria are
gram-positive, the color of bacteria under the microscope is purple and if the bacteria is
gram-negative, the color of bacteria under the microscope is red or pink color.
OBJECTIVES
1. To determine several techniques in microbiology.
2. To examine macroscopic/morphology and microscopic characteristics of Lactobacillus.

PROBLEM STATEMENT
Is the techniques in microbiology gives an impact to examine the characteristics of
Lactobacillus?

HYPOTHESIS
1. If we learn the techniques in microbiology, then we can examine the characteristics of
Lactobacillus
2. If the bacteria are gram-positive, the color of bacteria under the microscope is purple and
if the bacteria is gram-negative, the color of bacteria under the microscope is red or pink
color.
MATERIAL AND APPARATUS
1. Goat milk
2. Distilled water
3. Sterile water
4. MRS agar plate
5. Gram staining reagent (Crystal violet solution, iodine solution, acetone, and safranin)
6. Bunsen burner
7. Test tube
8. Vortex
9. Pipette
10. Hockey stick
11. Loop
12. Parafilm seal
13. Incubator
14. Slide
15. Tissue
16. Microscope
PROCEDURE

A. Serial Dilution Method

1. Sample milk was used for the experiment.
2. Each agar plate was labelled according to the concentration.
3. 1 ml of sample milk with 9 ml of distilled water was transferred to test tube.
4. Vortex was used to make it homogenous.
5. 1 ml of the liquid in test tube was transfer to another test tube until it reaches
concentration 106.

B. Spread Plate Method

1. 1 ml of each diluted sample was pipette from test tube to the spread plate.
2. Hockey stick was used to spread it evenly in spread plate.
3. The spread plate was incubated for 24 hours.

C. Streak Plate Method

1. The sterilize loop was heat and let cool down by stabbing it in the clean part of the agar.
2. A single colony was being pick up.
3. The colony was being strain in a new MRS agar plate.
4. The colony was being spread by using spread plate method.
5. Seal the plate with double layer parafilm.
6. The plate was being incubate at certain temperature for 24 to 48 hours.

D. Gram Staining
1. Distilled water was placed on slide by using sterile loop.
2. The colony of bacteria was being transferred to slide and heated gently.
3. A drop of crystal violet is added, and the slide was rinse with water.
4. Few drops of iodine were added to the slide. Then rinse with water.
5. The steps were repeated with acetone and safranin.
6. After the slide was dried, it was placed under the microscope.
RESULT

Image of Lactobacillus colonies on agar plate

1. Use table below to record your expected result on macroscopic/morphological
characteristic of Lactobacillus colonies.

Table 1.0: Macroscopic/Morphological results

MACROSCOPIC/MORPHOLOGICAL
OBSERVATION of Lactobacillus COLONIES

Margin/edges of Entire
the colonies

Shape of the colonies Circular

Chromogenesis/color of the colonies Creamy white

Surface of the colonies smooth

Texture of the colonies mucoid (sticky, mucus-like)

2. Use table below to record your expected result on microscopic characteristic of
Lactobacillus.

Table 2.0: Microscopic results

Image of Lactobacillus under light microscope

Bacteria gram type Gram-positive type

Shape of the bacteria Rod-shape

Magnification lens 100x magnification

DISCUSSION
Microbiology is the scientific study of microorganisms that we cannot see with our
naked eyes but can see using a microscope since microorganisms are minute living organisms
such as bacteria, archaea, algae, fungi, protozoa, and viruses (Pelczar, M. J. and Pelczar, Rita
M., 2020). Because of their different properties, certain bacteria have the ability to live and
grow in a certain range of environments, whereas others will perish or never grow in the
same environment. In this experiment, several techniques in microbiology, including the
serial dilution method, the spread plate method, the streak plate method, and the gram
staining method, are used to obtain a pure culture containing only one species or strain of
colony-forming organism. Bacteria are cultured on solid media and develop as colonies. A
colony is defined as a visible mass of microorganisms that all originate from a single mother
cell called a "colony forming unit" (CFU). Hence, a colony is a genetically identical clone of
bacteria ("Bacterial Colony Morphology," 2016).

For the serial dilution method, this method can estimate a sample's microorganism
concentration by counting the number of colonies cultured from serial dilutions of the sample
(Ben-David & Davidson, 2014). Goat milk was used as the sample to identify the desired
bacteria. Each agar plate was labelled according to the concentration. Then, 1 ml of sample
milk with 9 ml of distilled water was transferred to a test tube and a vortex was used to make
it homogenous. The liquid in the test tube was transferred to another test tube and the process
was repeated until the concentration reached a 10-6 dilution factor. The number of
microorganisms in a population can be determined using this technique. Furthermore, since
the pour plate method only counts the visible colony in enumeration, the maximum number
of organisms recovered is limited, whereas the determination of viable cells is necessary in
many microbiological procedures (Tankeshwar, 2021). Therefore, to simplify the procedure,
we approached the purpose of this serial dilution method, which is to reduce the
concentration of bacteria in a culture by a specific amount.

Next, the streak plate method is used to isolate a pure strain from a single
microorganism species, most often bacteria. The streak plate technique is a basic isolation
dilution procedure which main goal is to reduce the number of bacteria in each subsequent
quadrant (Aryal, 2021). Moreover, it is used to study the colony morphology of an organism.
By using a sterilized wire loop, the clean part of the agar was stabbed and a single colony was
picked up. The new media will only include organisms of a single species due to the use of
sterile (aseptic) techniques. The wire is then moved lightly along the agar surface, depositing
streaks of bacteria on the surface. The inoculating loop is flamed, and a few bacteria are
picked up from the 1st quadrant and streaked onto the 2nd quadrant. As the streaking
proceeds, fewer bacteria are deposited, and after each streak, the loop is flamed. After the 3rd
quadrant, individual organisms are deposited in the 4th quadrant, which is streaked last. For
this procedure, we used agar plates. Lactobacillus MRS (LMRS) agar is used as the medium
for this experiment to increase the growth of lactic acid bacteria (LAB) since MRS is a
selective medium that capable of supplying the micro- and macronutrients required for the
growth of Lactobacillus from milk sample (Rafieian-Kopaei et al., 2017). The agar plate was
then sealed with double-layered parafilm. After being incubated at a suitable temperature for
24 to 48 hours, small colonies appear. The colonies obtained are groups of offspring from
individual cells, and the streaking method is used to separate them. Individual cells are
separated and multiplied, thus forming discrete colonies, which are then multiplied to form
isolated colonies in the later quadrants (Tankeshwar, 2021).

The spread plate method is also used to isolate individual colonies from a diluted
sample of a mixed population. A successful spread plate should include visible and isolated
colonies of bacteria that are equally distributed and countable throughout the plate (Batra,
2018). Following incubation, the colonies on the agar plate was observed where some of the
colonies will be separated from one another. One of the colony was selected from the plate
and the elevation, pigmentation, and size was recorded. This approach is used to investigate
the cultural properties of the specimen and to isolate bacteria in discrete colonies from a
sample containing more than one bacterium (Batra, 2018). It is critical to ensure that some of
the cells in the specimen or diluted specimen are separated from each other by a sufficient
distance to allow the colonies that form to be free of each other (Hartman D, 2011). Therefore,
the experiment consisted of accurately measuring the quantity of the specimen when
preparing serial dilutions, measuring the quantity of the diluted specimen while inoculating
onto the solidified media plates, and uniformly spreading the specimen onto the media plate
to obtain discrete and well-developed colonies. The number of colonies in the cultures media
plates inoculated with serial dilutions of the specimen will get smaller and smaller as the
dilution factor goes up. This means that the highest number of colonies will grow in the first
plate and the least number of colonies will grow in the last plate (Hartman D, 2011).
 From the results obtained, the macroscopic or morphological characteristics of
Lactobacillus colonies can be analysed. These methods are used to study the colony
morphology of an organism (Tankeshwar, 2021). Edge, shape, elevation, colour, and texture
are all characteristics of a colony that are analysed to help identify bacteria. It can be seen
that the margin (edge) of Lactobacillus colonies is entire. They are circular in shape and
appear creamy white in colour. The colonies also have a smooth surface and a mucoid texture,
which is a sticky, mucus-like texture.

One of the most important staining techniques in microbiology is Gram staining. The
basic principle of gram staining involves the ability of the bacterial cell wall to retain the
crystal violet dye during solvent treatment.Gram-positive organisms are organisms that
retain their primary colour and appear purple-blue when viewed under a microscope while
gram-negative organisms appear red under a microscope when they do not take up primary
stain(Tripathi, N., Sapra, A. 2021). Gram-positive microorganisms have higher
peptidoglycan content, whereas gram-negative organisms have higher lipid content (Tripathi,
N., Sapra, A. 2021). Gram-negative bacteria have an outer lipid membrane, while Gram-
positive bacteria do not. As a resut, it is easy to penetrate the violet dye for gram positive
compared to gram-negative bacteria which it has outer lipid membrane as extra protection.
The result of experiment indicates that Lactobacillus spp. are facultatively anaerobic,
catalase-negative, Gram-positive, non-spore-forming rods that often grow better under
microaerophilic conditions (Goldstein et al., 2015). Their gram stain morphology can vary,
including as short, plump rods, long, slender rods, in chains or palisades (Goldstein et al.,
2015). Lactobacillus is gram-positive bacteria appear purple-blue because their thick
peptidoglycan membrane can hold the dye . Thus, Lactobacillus spp. is called gram-positive
due to the positive result.
Post-Lab Questions:

1. List down the microbiological techniques in isolation of Lactobacillus involved in this

video.
1.1. Serial dilution method
1.2. Spread plate method
1.3. Streak plate method
1.4. Gram staining

2. What kind of agar medium used to isolate the Lactobacillus in this video?
Lactobacillus MRS (LMRS)

3. What is the purpose of using that type of agar medium?

The purpose is to increase the growth of lactic acid bacteria (LAB) since it is a selective
medium that capable of supplying the micro- and macronutrients required for the growth
of Lactobacillus from the milk sample.

4. What are other possible bacteria can be found in dairy products?

Streptococcus, Lactococcus, Bifidobacteria, Entrococcus and Pediococci.

5. Explain the aseptic technique used in this video.

Successful cell culture is primarily reliant on maintaining the cells free of microbes such
as bacteria, fungus, and viruses. The aseptic technique, which is intended to form a barrier
between microorganisms in the environment and the sterile cell culture. According to the
video, physical methods are utilised to avoid contamination, with heat being the most
common. Heat kills microbes via denaturing protein, melting lipids, and incineration
when used with an open flame. To avoid contamination of a culture, flame is used to
sterilise inoculating loops and the mouths of culture tubes. Also, the plate was incubated
upside down at the appropriate temperature in an incubator, which is used to sterilise
objects that would be damaged by moisture exposure usually for 1 to 2 hours at a
temperature of 160°C.
CONCLUSION

In conclusion, the types of bacteria that exist in the Local Dairy which is goat milk in
this experiment, are both Gram’s positive and Gram’s negative bacteria. The inoculation of
bacteria from Local Dairy by streaking method is to determine the type and the isolation of
bacteria. As goat milk contain and Probiotic Lactobacillus, this method also to determine the
presence of bacteria according to its shape and color after Gram Staining. As the bacteria has
its own unique shape, it is easily can be determine under the microscope. The goat milk will
be leave for a week then the result and the condition of the yogurt will be observed.

Serial dilution is the sequential on dilution used to reduce the dense culture of cells or
sample to a more usable concentration. Serial dilution also used to accurately create high
diluted solution. CFU is the method to calculate the number of isolated bacteria in the colony.

Gram Staining is the method to identify and determine the type of bacteria that exist.
There are two types of Gram’s bacteria which is Gram’s positive bacteria and Gram’s
negative bacteria. For Gram’s positive bacteria the color will turn pink or red after the crystal
violet solution and Lugol’s iodine solution are added into it. This is because of the layer of
peptidoglycan are thin. While the Gram’s negative bacteria will remain purple after crystal
violet solution and Lugol’s iodine solution are added. The layer of the peptidoglycan for the
Gram’s negative bacteria is thick.
REFERENCES

Aryal, S. (2021, July 8). Retrieved from Streak Plate Method- Principle, Methods,
Significance, Limitations: https://microbenotes.com/streak-plate-method-principle-
methods-significance-limitations/
Bacterial Colony Morphology. (2016, March 26). Retrieved January 11, 2022, from Biology
LibreTexts website:
https://bio.libretexts.org/Learning_Objects/Laboratory_Experiments/Microbiology_La
bs/Microbiology_Labs_I/08%3A_Bacterial_Colony_Morphology

Batra, S. (2018). Spread Plate Technique For the Isolation of Microorganism | Culture
Methods in Microbiology Laboratory. Paramedics World.
https://paramedicsworld.com/microbiology-culture-techniques/spread-plate-culture-
method-isolation-bacteria-microorganism-pure-culture/medical-paramedical-
studynotes#.XGFykFwzbIU

Ben-David, A., & Davidson, C. E. (2014). Estimation method for serial dilution experiments.
Journal of Microbiological Methods, 107, 214–221.
https://doi.org/10.1016/j.mimet.2014.08.023

Goldstein, E. J. C., Tyrrell, K. L., & Citron, D. M. (2015). Lactobacillus Species: Taxonomic
Complexity and Controversial Susceptibilities. Clinical Infectious Diseases,
60(suppl_2), S98–S107. https://doi.org/10.1093/cid/civ072

Hartman D. (2011). Perfecting your spread plate technique. Journal of microbiology &
biology education, 12(2), 204-5. doi:10.1128/jmbe.v12i2.324

Kim, H., Kim, T., Kang, J., Kim, Y., & Kim, H. (2020). Is Lactobacillus Gram-Positive? A
Case Study of Lactobacillus iners. Microorganisms, 8(7), 969.
https://doi.org/10.3390/microorganisms8070969

Pelczar, M. J. and Pelczar, . Rita M. (2020, December 4). microbiology. Encyclopedia

Britannica. https://www.britannica.com/science/microbiology
Rafieian-Kopaei, M., Karami, S., Roayaei, M., Hamzavi, H., Bahmani, M., Hassanzad-Azar,
H., & Leila, M. (2017). Isolation and identification of probiotic Lactobacillus from
local dairy and evaluating their antagonistic effect on pathogens. International Journal
of Pharmaceutical Investigation, 7(3), 137. https://doi.org/10.4103/jphi.jphi_8_17

Tankeshwar, A. (2021, June 3). Retrieved from Streak Plate Method: Principle, Procedure,
Uses: https://microbeonline.com/streak-plate-method-principle-purpose-procedure-
results/

Tankeshwar, A. (2021, June 11). Retrieved from Pour Plate Method: Procedure, Uses, (Dis)
Advantages: https://microbeonline.com/pour-plate-method-principle-procedure-uses-
dis-advantages/

Tripathi, N., & Sapra, A. (2021). Gram Staining. In StatPearls. StatPearls Publishing.

Sagar, A.(2018). Gram Staining: Principle, Procedure, Interpretation, Examples and

Animation. https://microbiologyinfo.com/gram-staining-principle-procedure-interpretation-
examples-and-animation/
EXPERIMENT 2: TECHNIQUE IN DNA TECHNOLOGY

INTRODUCTION
DNA technology has revolutionized science (Khan et al., 2016). DNA, or an
organism's genetic material, holds many clues that have unlocked some of the mysteries
behind human behaviour, disease, evolution, and ageing. DNA-based technologies will
emerge as technology advances our understanding of DNA. DNA extraction, Polymerase
Chain Reaction (PCR) and Gel electrophoresis are some examples of DNA technology.
Furthermore, DNA technologies are already shaping medicine, forensics and environmental
science as well as national security (Cobb, n.d.).

DNA extraction is the removal of deoxyribonucleic acid (DNA) from the cells of the
sample. The fundamental of DNA extraction is lysis the cell containing the DNA of interest.
There are physical methods and chemical methods. Physical methods use bead beating,
French pressure cell press, sonicating or manual grinding with liquid nitrogen such as mortar
and pestle method. The chemical method which is broken down proteinaceous cellular wall
by vortexing with phenol and centrifuged the protein will remain in the organic phase. It is
essential to add a detergent Sodium Dodecyl Sulfate (SDS) to remove the lipid component of
the cell membrane. After that, precipitate the protein by addition of a salt such as ammonium,
sodium and potassium acetate. Then, DNA of interest is precipitate by mixing with the cold
ethanol or isopropanol then spin it by using centrifuge (Rice, 2019). The DNA is insoluble
and come out of the solution. The alcohol is use to remove the salt previously added and the
resultant DNA pellet had washed with cold alcohol again. After drying, the DNA can be re-
suspended in a buffer such as Tris and TE. The presence of DNA then confirmed using
electrophoresing on an agarose gel containing ethidium bromine or another fluorescent dye
that react with the DNA and checking under UV light.

Next, Polymerase Chain Reaction (PCR) function to synthesize a huge number of

copies of gene. The PCR comprises of three major steps which is denaturation, annealing
and extension that repeated up to 40 cycles. This reaction is performed on an automated
cycler called PCR machine designed to heat and cool the tubes with the reaction mixture in
programmed time intervals. The starting materials for PCR are double-stranded DNA
containing target nucleotide sequence to be copied, a heat- resistance DNA polymerase, all
four nucleotides and two short single stranded DNA molecules that serve as primers in PCR
technique (Cui et al., 2008). One primer is complementary to one end of the target sequence,
the second is complementary to the other strand at the other end of the sequence.
 Then, Gel electrophoresis is technique of separation of DNA fragments according to
their size based on movement through a gel medium when an electric field is applied (Isbir et
al., 2013). In this technique, a gel box is used to separated DNA in an agarose gel with an
electrical charge. DNA is a negatively charge molecule due to phosphate group in their sugar-
phosphate backbone. The DNA will move through the gel toward the positive charge when
the red and black leads are plugged into a power supply. DNA move at different rates
depends on their size, the larger piece moving more slowly through the porous medium,
hence creating a size separation that can be differentiated in a gel (Isbir et al., 2013). Lastly,
the size of PCR DNA bands can be measured by comparing them to a DNA ladder or marker.

OBJECTIVES

1. To study the technique in DNA extraction, PCR and Gel Electrophoresis

2. To estimates the sizes of DNA band from the PCR reaction

PROBLEM STATEMENT

1. How does the study of technique in DNA extraction, PCR and Gel Electrophoresis differ
from each other?
2. How does the PCR technique estimate the sizes of DNA band?

HYPOTHESES

The sizes of DNA band from the PCR reaction can get by comparing the bands in a sample to
the DNA marker.
MATERIALS & APPARATUS

Practical 2.1: Plasmid DNA Isolation Using Alkaline Lysis Method

1. Bacteria culture (Escherichia coli culture)

2. 1.5 mL microcentrifuge tubes
3. Centrifuge
4. Micropipette and tips (1000µL, 200µL, 20µL)
5. Incubator shaker
6. -20°C freezer
7. Glucose
8. Tris-Cl
9. Ethylene diamine tetraacetic acid (EDTA)
10. M Sodium hydroxide (NaOH)
11. Sodium dodecyl sulphate (SDS)
12. Potassium acetate
13. Glacial acetic acid
14. sdH2O
15. Isopropanol
16. Ethanol 1 mL, 70%v/v (cold)
17. Nutrient broth media
18. Dry ice or ice cubes
19. RNase
20. 50 µL of 0.1 x TE buffer - Tris/EDTA (TE)
21. Solution I (Glucose 50mM; Tris-Cl 25mM; EDTA 10mM)
22. Solution II (NaOH 0.2N; SDS 1%w/v)
23. Solution III (Potassium acetate 5M, glacial acetic acid)
Practical 2.2: Agarose Gel Electrophoresis

1. Plasmid extract (from practical 2.1)

2. Micropipette and tips (1000µL, 200µL, 20µL)
3. Gel electrophoresis set (gel casting tray, comb) and power supply
4. UV transilluminator
5. Latex glove
6. Microwave
7. Hot plate
8. Gel tank
9. Gel safe stain
10. DNA stain (Ethidium bromide/ EtBr)
11. 100 mL of 1 x TBE buffer - Tris/Borate/EDTA (TBE)
12. RNase
13. Dry ice or ice cubes
14. DNA ladder-loading dye mixture (10 µL 1 kb DNA ladder + 2 µL loading dye)
15. DNA sample-loading dye mixture (10 µL extracted plasmid DNA + 2 µL loading dye)
16. DNA marker 1 kb
17. Agarose powder
18. 70% ethanol
PROCEDURE

Practical 2.1: Plasmid DNA Isolation Using Alkaline Lysis Method

1. 5mL of bacterial broth culture was prepared in a 50mL centrifuge tube.

2. The tube was centrifuged at 12000 rpm for 1 minute at room temperature and the pellet
was formed.

3. The supernatant (media broth) was poured off immediately, and the undisturbed pellet
(bacterial cells) was left behind at the bottom of the tube.

4. The pellet was resuspended with 100 µL of ice-cold solution I (a resuspension buffer
containing glucose, Tris, & EDTA). The pellet was made sure to be resuspended
completely by gently tapping the tube until no cell clumps remained. The tube was left for
5 minutes at room temperature.

5. 200 µL of freshly prepared solution II (a lysis buffer containing SDS, NaOH) was added
into the tube. The solution was mixed by gently inverting (5 times) the tube. The tube was
left in ice for 10 minutes. The mixture became viscous as the bacteria burst open and their
contents leaked into the solution.

6. 200 µL of solution III (a neutralization solution containing potassium acetate, glacial

acetic acid) was added into the tube. The solution was mixed by gently inverting (5 times)
the tube. The tube was left in ice for 10 minutes. A fluffy white precipitate was formed.

7. The tube was centrifuged at 12000 rpm for 5 minutes (the load was made sure to be
balanced).

8. The supernatant (containing plasmid DNA) was transferred into a new centrifuge tube.

9. 300 µL of ice-cold isopropanol was added into the tube. The solution was mixed gently
by tapping the tube. The tube was kept in the freezer (-20°C) for 10 minutes. Plasmid
DNA and RNA were precipitated out of the solution.
10. The tube was centrifuged at 12000 rpm for 5 minutes.

11. The supernatant (isopropanol) was discarded and the pellet (precipitated plasmid DNA)
was left in the tube. The excess liquid (supernatant) was blotted from the pellet using a
clean tissue paper.

12. 1 mL of 70% ethanol was added into the tube to wash the plasmid DNA pellet. The
solution was mixed gently by tapping the tube. The tube was centrifuged at 12000 rpm for
10 minutes.

13. The supernatant (ethanol) was discarded and the pellet (washed precipitated DNA) was
left in the tube. The pellet was air dried for 10 minutes.

14. The plasmid DNA pellet was re-dissolved in 50 µL of 0.1 x TE buffer.

Practical 2.2: Agarose Gel Electrophoresis

1. The electrophoresis apparatus (gel casting tray, comb) was cleaned with 70% ethanol.

2. To prepare a 1% w/v agarose gel, 1 g of agarose powder was added in 100 mL of 1 x

TBE buffer.

3. The agarose was melted in a microwave until the agarose was completely dissolved.

4. The solution was cool down for 5 minutes.

5. 1 µL of DNA stain (Ethidium bromide/EtBr) was added to the cooled agarose solution.
The solution was swirled to mix well.

6. The comb was placed in the gel casting tray.

7. The agarose solution was poured into the casting tray.

8. The agarose solution was allowed to cool down until it is solidified.

9. The gel tank was filled with 1 x TBE buffer.

10. Once the agarose gel is solidified, the comb was removed carefully.

11. The gel with the tray was placed in the gel tank. The gel was made sure to be completely
covered with 1 x TBE buffer (about 2-3 mm of buffer over the gel).

12. 12 µL of “DNA ladder-loading dye mixture” (10 µL 1 kb DNA ladder + 2 µL loading dye)
was carefully pipetted into the first well of the gel.

13. 12 µL of “DNA sample-loading dye mixture” (10 µL extracted plasmid DNA + 2 µL

loading dye) was carefully pipetted into the additional wells of the gel. The samples were
loaded into the wells using a micropipette and was taken care not to mix samples between
wells.
14. The electrode wires were connected to the power supply and electrophoresis was
performed at 100 V for 30 minutes.

15. After the electrophoresis was completed, the power supply was switched off. The
electrodes were disconnected from the power supply. The gel with the tray was taken out
from the gel tank.

16. The gel was analysed. A device that has UV light was used (UV transilluminator), the
separated was visualized and DNA samples/DNA ladder were stained on the gel.

17. The DNA ladder in the first lane was used as a guide. The bands that were detected in the
sample lanes were interpreted to determine the presence or absence of DNA samples and
the size (length of DNA molecule) of the product was quantified.
RESULTS
Use the image of Gel electrophoresis below to determine the sizes of the DNA band
which is the products from DNA extraction and PCR.

Image 1.0: Gel Electrophoresis of DNA sample

DISCUSSION
Gene is a segment of DNA that contain the message to encode information to produce protein.
Genes have four types which is Adenine (A) Thymine (T), Cytosine (C) and Guanine (G).
DNA is a molecule that contain information an organism needs to develop. DNA is made
from nucleotides. These building blocks are made of three parts: a phosphate group, a sugar
group and one of four types of nitrogen bases. DNA has a negatively charged which is at
phosphate group. If it was placed in electric field, it will move from negative pole to positive
pole. During gel electrophoresis, DNA is placed in gel box agarose gel. When electric field
occur in the gel box, the DNA will migrate through the gel towards the positive charge due to
the net negative charge pf the DNA molecule. During gel electrophoresis the smaller the
DNA pieces move faster and farther through the gel while the larger pieces of DNA moves
slower and near the gel.

The first ladder in the image 1.0 is DNA marker, it produces different DNA band with known
sizes. We can estimate the size of DNA by referring the marker. DNA 1 have two band with
estimated size 200bp to 100 bp respectively. DNA 2 has two band with estimated size 275 bp
to 65 bp respectively. DNA 3 has 3 DNA band with estimated size 275 bp,200 bp and 90 bp
respectively. DNA 4 has three band with size 275 bp, 200 bp and 90 bp respectively. DNA 5
has two band with size 200 bp and 65 bp respectively. DNA 6 has three band with size 275bp,
90 bp and 65 bp respectively.

PCR have created many copies of same DNA. This can be proved from the gel
electrophoresis image where most of the DNA have similar band size between 275 bp to 65
bp. Based from the DNA ladder the smallest size is 50 bp. It indicates that it has a small DNA
piece. The smaller the DNA piece the farther it will move in gel box. Most of the DNA
sample has a small DNA size.

This experiment consists of three method which are DNA extraction, PCR, and gel
electrophoresis. Some precautions steps are do not disturb the precipitate while discard the
supernatant during DNA extraction. Besides that, micropipette must be changed after using
for one sample. This is important to get better results.
Post Lab Question

1. By referring to the Image 1.0, estimate the size of DNA fragments in the yellow circle.
From the image 1.0 the size of DNA band in yellow circle for DNA 4 is 200 bp. While
for DNA 6, it has 3 DNA band which are 50bp, 100bp and 250bp respectively.

2. What is your assumption on DNA samples from this gel electrophoresis image? (put the
answer in discussion)

3. What is the purpose of DNA extraction, PCR and Gel electrophoresis?

DNA extraction is used to remove the deoxyribonucleic acid (DNA) from cell of sample.
Polymerase chain reaction (PCR) is a method to make multiple copies of a segment DNA.
Gel electrophoresis is a laboratory method used to separate mixtures of DNA, RNA, or
proteins according to molecular size. In gel electrophoresis, the molecules to be separated
are pushed by an electrical field through a gel that contains small pores.

4. Why is the DNA samples and all the processes must be kept in cold ice?
During DNA extraction, the DNA sample and all other processes must be kept in ice cold
because it will increase yield of DNA. Cold condition can protect the DNA from break it
apart by slowing the enzyme that acts on them.

5. Why is changing the tips of micropipette is important during the DNA technology
processes?
To avoid an aerosol or liquid residue from one sample carried over to the next, which is
known as sample-to-sample contamination (or carry-over contamination). For example,
this can happen when the same pipette tips are used several times.

6. What is the function of these reagents in PCR process (refer the video)?
a. Forward primer
The forward primer attaches to the start codon of the template DNA (the anti-sense
strand).
 b. Reverse primer
The reverse primer attaches to the stop codon of the complementary strand of DNA (the
sense strand).

c. RedTAq
To make PCR more convenient by formulating a reaction that included both loading
buffer and dye. The final product would allow the researcher to know whether samples
included enzyme and would eliminate one step in post-PCR product analysis, but it would
not hinder any potential post-PCR applications for the amplified DNA.

7. In Gel Electrophoresis process (refer the video):

a. What is the function of loading dye?
Loading dyes give colour to the samples, making the loading process easier to see.
Besides, the loading dyes improve the sample's density, ensuring even loading in the
sample well.

b. What is the function of DNA ladder?

DNA ladder allows you to estimate the size of the unknown fragment by comparing it to
the closest band in the ladder lane.

c. How many volumes of DNA sample being loaded in the gel electrophoresis well?
3 μL of the sample were being loaded in the gel electrophoresis well.
CONCLUSION

In conclusion, we studied the methodologies of DNA extraction, PCR, and gel

electrophoresis. DNA extraction is the process of removing deoxyribonucleic acid DNA from
a sample, which must be kept on ice to maximize DNA yield and prevent DNA fragmentation.
The polymerase chain reaction (PCR) then replicates a segment of DNA by exponentially
amplifying copies of extremely short DNA sequences across a series of temperature changes.
Moreover, gel electrophoresis separates mixtures of DNA, RNA, or proteins according to
molecular size pushed by an electrical field through a gel that contains small pores at a speed
that is inversely related to their lengths. As a result, a small DNA molecule will move further
through the gel than a larger DNA molecule. Finally, by comparing the bands in a sample to
the DNA ladder or DNA marker, we can determine the sizes of DNA bands from PCR
reactions.
REFERENCES

Cobb, B. (n.d.). DNA Technology | Encyclopedia.com. Www.encyclopedia.com.

https://www.encyclopedia.com/science/encyclopedias-almanacs-transcripts-and-

maps/dna-technology

Cui, G., Qin, L., Wang, Y., & Zhang, X. (2008). An encryption scheme using DNA

technology. 2008 3rd International Conference on Bio-Inspired Computing: Theories

and Applications. https://doi.org/10.1109/bicta.2008.4656701

Isbir, T., Kirac, D., Demircan, B., & Dalan, B. (2013, January 1). Gel Electrophoresis (S.

Maloy & K. Hughes, Eds.). ScienceDirect; Academic Press.

https://www.sciencedirect.com/science/article/pii/B9780123749840005805

Khan, S., Ullah, M. W., Siddique, R., Nabi, G., Manan, S., Yousaf, M., & Hou, H. (2016).

Role of Recombinant DNA Technology to Improve Life. International Journal of

Genomics, 2016, 1–14. https://doi.org/10.1155/2016/2405954

Rice, G. (2019). DNA Extraction. Genomics.

https://serc.carleton.edu/microbelife/research_methods/genomics/dnaext.html