Report on Molecular Laboratory Practices at Hawassa University Agricultural

Campus

Introduction

Molecular biology laboratories are integral to biotechnology, enabling the study of cellular and
genetic materials at the molecular level. These facilities are essential for advancing research in
genetics, diagnostics, and therapeutics. The molecular biology laboratory at Hawassa University
Agricultural Campus supports students and researchers with state-of-the-art equipment and
protocols for various applications, including DNA extraction and purification. This report
outlines the components of a molecular laboratory, the detailed DNA extraction and purification
procedure, its objectives, and the associated benefits.
Central biotechnology laboratory sponsored by Norwegian government on the agricultural and
plant science Hawassa University.

Objectives

The objectives of this report and the associated laboratory practices include:

1. To document the DNA extraction and purification procedure in a molecular biology

laboratory, ensuring clarity and reproducibility.
2. To provide an understanding of the tools, reagents, and protocols used in molecular
laboratories at Hawassa University Agricultural Campus.
3. To highlight the significance of DNA extraction techniques in research, diagnostics, and
biotechnological applications.
4. To equip biotechnology students with the theoretical and practical knowledge required to
perform molecular biology experiments accurately.
5. To emphasize the importance of standardized protocols in ensuring the reliability and
quality of experimental outcomes.

Description of Activities Carried Out During the Internship

• Overview

Components of a Molecular Laboratory

A molecular biology laboratory comprises specialized tools and reagents designed to ensure
accuracy, reliability, and sterility in experiments. The primary components include:

1. Essential Equipment:
o Micro centrifuges
o Thermal cyclers (PCR machines)
o Gel electrophoresis apparatus
o Spectrophotometers
o Micropipettes and tips
o Water baths

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 2. Reagents and Consumables:
o DNA extraction kits
o Buffers (e.g., lysis buffer, washing buffer, elution buffer)
o Protease enzymes
o Ethanol and isopropanol
o Nuclease-free tubes and pipette tips
3. Safety and Sterility Supplies:
o Biosafety cabinets
o Gloves and lab coats
o Disinfectants and autoclaves
4. Data Analysis Resources:
o Computers equipped with bioinformatics software

DNA extraction

DNA extraction is a fundamental technique in molecular biology that involves isolating

deoxyribonucleic acid (DNA) from cells or tissues. This process is critical for studying genetic
material, conducting diagnostic tests, and enabling biotechnological applications such as cloning,
sequencing, and genome editing. Extracting high-quality DNA is essential for ensuring the
success of downstream applications, as contaminants can interfere with enzymatic reactions and
analytical processes. The procedure described in this report is optimized for extracting DNA
from blood and body fluids, ensuring reliable results for a variety of research and diagnostic
purposes.

Objective

The primary objective is to provide a formal documentation of the DNA extraction and
purification procedure performed at Hawassa University Agricultural Campus. This includes a
step-by-step account of the optimized protocol using passive past tense to ensure clarity and
formality.

DNA Extraction and Purification Procedure

The DNA extraction and purification protocol was optimized for frozen blood samples up to 250
µL in volume. It was also applicable to anti-coagulated blood, saliva, serum, and buffy coat. The
steps were carried out as follows:

1. The sample was transferred into a sterile micro centrifuge tube, and the volume was
adjusted to 250 µL using 10 mM Elution Buffer.
2. 25 µL of OB Protease solution and 250 µL of BL Buffer were added. The mixture was
vortexed at maximum speed for 15 seconds.
3. The mixture was incubated at 65°C for 10 minutes, with brief vortexing during the
incubation.
4. 260 µL of 99% ethanol was added, and the mixture was vortexed at maximum speed for
20 seconds.
5. The tube was centrifuged briefly to collect any droplets from the inside of the lid.

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6. A HiBind DNA Mini Column was inserted into a 2 mL Collection Tube.
7. The entire sample was transferred to the column.
8. The column was centrifuged at ≥ 10,000 x g for 1 minute.
9. The filtrate and the collection tube were discarded.
10. The HiBind DNA Mini Column was inserted into a new 2 mL Collection Tube.
11. 500 µL of HBC Buffer diluted with 95-99% isopropanol was added. The column was
centrifuged at ≥ 10,000 x g for 1 minute, and the filtrate was discarded. The collection
tube was reused.
12. 700 µL of DNA Wash Buffer diluted with 99% ethanol was added. The column was
centrifuged at ≥ 10,000 x g for 1 minute, and the filtrate was discarded. The collection
tube was reused.
13. Step 12 was repeated for a second DNA Wash Buffer wash.
14. The empty HiBind DNA Mini Column was centrifuged at maximum speed for 2 minutes
to dry the column. This step was critical for the removal of trace ethanol.
15. The HiBind DNA Mini Column was transferred into a nuclease-free 2 mL micro
centrifuge tube.
16. 100-200 µL of Elution Buffer heated to 65°C was added. The column was left at room
temperature for 5 minutes before centrifugation at maximum speed for 1 minute.
17. Step 16 was repeated for a second elution step.
18. The eluted DNA was stored at -20°C.

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Benefits of DNA Extraction Knowledge

1. Facilitates Research: Enables the study of genetic material for advancements in gene
function analysis and trait mapping.
2. Supports Diagnostics: Provides purified DNA for diagnostic applications, including
disease identification and pathogen detection.
3. Enhances Biotechnological Applications: Enables processes such as cloning,
sequencing, and genome editing.
4. Develops Technical Skills: Improves proficiency in handling molecular techniques,
crucial for careers in biotechnology and research.
5. Promotes Quality Control: Ensures reproducibility and reliability in molecular
experiments by adhering to standardized protocols.

Conclusion

The DNA extraction and purification protocol is a fundamental technique in molecular biology,
providing high-quality DNA essential for downstream applications. At Hawassa University
Agricultural Campus, adherence to such protocols ensures the reliability and reproducibility of
results. Mastery of these techniques equips biotechnology students with the necessary skills to
contribute effectively to research and industry advancements.

Over view of lab equipment and use

Vertical Electrophoresis

 Purpose: Used to separate proteins or small DNA

fragments.
 How It Works:
o Proteins or DNA samples are loaded into vertical
gels (usually polyacrylamide).
o An electric current separates molecules based on
their size.
 Common Use: Protein analysis (e.g., SDS-PAGE for
molecular weight determination).

Horizontal Gel Electrophoresis

 Purpose: Used to separate larger DNA or RNA

molecules.
 How It Works:
o DNA or RNA samples are loaded into horizontal gels (usually agarose).
o An electric current separates molecules based on size.
 Common Use: DNA analysis (e.g., checking PCR products, restriction digestion).

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Mortar Mill

A mortar mill is a laboratory or industrial tool used for

grinding, mixing, and homogenizing solid or semi-solid
materials. It typically consists of a mortar (a bowl-like
container) and a rotating pestle or grinding arm.

Uses of Mortar Mill

1. Grinding and Crushing:

o Used to grind solid samples into fine powders.
o Commonly used for grinding minerals, rocks,
and other hard materials.
Mixing

Colony Counter
A colony counter is a laboratory device used to count colonies of microorganisms (such as
bacteria or fungi) growing on a solid medium, typically in petri dishes.

Uses of a Colony Counter

1. Counting Microbial Colonies:

o Used to count the number of distinct colonies
on agar plates.
o Helps quantify microbial growth after incubation.
2. Estimating Microbial Load:
o Determines the concentration of microorganisms in
a sample (e.g., water, food, soil, or clinical
specimens).
o Commonly used in microbial quantification in CFU
(Colony Forming Units).
3. Quality Control:
o Monitors contamination levels in food,
pharmaceuticals, or other products.
o Ensures compliance with hygiene and safety
standards.

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pH Meter
A pH meter is a scientific instrument used to measure the acidity or alkalinity (pH) of a liquid or
semi-solid sample. It provides a numeric value representing how acidic or basic a solution is, on
a scale from 0 to 14.

Uses of a pH Meter

1. Measuring pH:
o Determines the hydrogen ion concentration in a
solution.
o pH < 7 indicates acidity, pH = 7 is neutral, and pH
> 7 indicates alkalinity.
2. Quality Control:
o Ensures the pH of products like beverages,
cosmetics, or pharmaceuticals meets standards.
o Monitors pH in industrial processes (e.g., chemical
production).

Vertical gel electrophoresis apparatus

This device is a vertical gel electrophoresis apparatus,

commonly used in molecular biology and biochemistry labs to
separate and analyze proteins or small DNA fragments.

Key Features:

 The gel is set in a vertical orientation.

 Includes electrodes (positive and negative terminals) for
applying an electric current.
 Typically used with polyacrylamide gels (e.g., for
SDS-PAGE).

Purpose:

 Protein Analysis: To separate proteins based on their

size.
 DNA Analysis: To separate small DNA fragments.

Safety Cabinet or Hood

A safety cabinet or hood is a specialized enclosure used in laboratories to provide protection

during work with hazardous materials, including chemicals, microorganisms, or particles. It
ensures the safety of the user, the environment, and sometimes the materials being handled.

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Types and Uses of Safety Cabinets or Hoods

1. Biological Safety Cabinet (BSC):

o Purpose: Protects the user, the environment, and biological samples from
contamination.
o Uses:
 Handling pathogenic microorganisms (bacteria,
viruses).
 Cell culture work.
 Research in microbiology or molecular biology.
2. Chemical Fume Hood:

Purpose: Protects the user from harmful chemical fumes,

vapors, or dust.

Uses:

 Working with volatile or toxic chemicals.

 Mixing chemicals or performing reactions.

Bacterial Colony Counter

A bacterial colony counter is a laboratory device used to count bacterial colonies grown on
agar plates after incubation. It helps quantify the number of bacteria in a sample by measuring
the Colony-Forming Units (CFU).

Uses of a Bacterial Colony Counter

1. Counting Colonies:
o Quickly and accurately counts bacterial colonies on
petri dishes.
o Reduces human error during manual counting.
2. Microbial Quantification:
o Helps determine the concentration of bacteria in a
sample.
o Results are expressed in CFU/mL or CFU/g,
depending on the sample type.
3. Quality Control:
o Ensures microbial limits are within acceptable
ranges in food, beverages, cosmetics, and
pharmaceuticals.
o Detects contamination in industrial or clinical samples.

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3D shaker

A 3D shaker, also known as a 3D orbital shaker or three-

dimensional shaker, is a laboratory instrument used for
mixing, shaking, or agitating liquids, solids, or a combination
of both. The 3D shaker moves in multiple directions, typically
along the x, y, and z axes, providing a more thorough and
uniform mixing action compared to traditional shaking
methods. This movement is typically achieved using an
oscillating or rotating platform that ensures the sample moves
in a three-dimensional motion.

Key Features and Uses:

1. Uniform Mixing: The 3D motion helps achieve a more homogeneous mix of samples,
which is crucial for certain experiments.
2. Cell Culture: It is commonly used in biology and biotechnology laboratories, especially
for culturing cells and microorganisms. The gentle mixing ensures optimal growth
conditions without damaging the samples.

Pipette controller

A pipette controller is a laboratory tool used to assist in the precise

and efficient transfer of liquids using a pipette. It is designed to make
the process of drawing liquid into and dispensing it from a pipette
easier and more controlled, especially when working with larger
volumes or when accuracy is critical.

Key Features and Functions:

1. Enhances Precision and Control: The pipette controller is

designed to provide a steady, controlled suction and
dispensing of liquid, ensuring accurate transfer of small
volumes. This is particularly helpful when using glass
pipettes, which are typically more fragile and require a
delicate touch.
2. Ease of Use: It allows the user to draw up and dispense liquid
by simply pressing buttons or using a finger-operated
mechanism. The controller usually features a bulb or
motorized pump that generates suction for the pipette.

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Scientific centrifuge

Scientific centrifuge is a laboratory instrument

used to separate components of a mixture
based on their different densities by applying
centrifugal force. It is commonly used in
various scientific fields, such as biology,
chemistry, medicine, and environmental
science, for separating liquids, precipitates,
cells, proteins, and other substances.

Key Features and Functions:

 Centrifugal Force: The centrifuge

works by rapidly spinning a sample in a
tube or container at high speeds. This
motion creates a force that pushes particles away from the center of rotation, causing
denser substances to move toward the bottom (forming a pellet) while less dense
components remain at the top (supernatant).

Dispenser

A dispenser in a laboratory context is a device used to accurately and

consistently dispense liquids or powders from a larger container into
smaller, controlled amounts. Dispensers help improve the precision and
efficiency of tasks such as reagent preparation, sample handling, and
precise liquid transfer in experiments.

Key Features and Functions:

 Precise and Controlled Dispensing: Dispensers are designed to

release specific volumes of liquids or powders. This ensures
accuracy, which is crucial for experiments that require exact
amounts of reagents or solutions.

Digital balance

A digital balance is a laboratory instrument used to measure the mass of an object or substance
with high precision and accuracy. Unlike traditional mechanical balances, which use a system of
beams and weights, a digital balance employs electronic sensors and displays to give a direct
reading of the mass, typically in grams, milligrams, or other units.

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Key Features and Functions:

 Precision and Accuracy: Digital balances offer high

precision, often to the milligram (mg) or even microgram
(µg) level, making them essential for tasks where exact
measurements are critical. The balance measures mass by
determining the force exerted by the object due to gravity
and converting this into a readable value.

GelDoc system

A GelDoc system (short for Gel Documentation system) is a laboratory instrument used for
imaging and analyzing gels that are used in molecular biology and biochemistry. This system is
essential for visualizing and documenting results from techniques like electrophoresis, which is
used for the separation of biomolecules such as DNA, RNA, and proteins based on their size,
charge, and other properties.

Key Features and Functions:

1. Gel Imaging: The primary purpose of a GelDoc system is to capture high-quality images
of gels, such as agarose or polyacrylamide gels that
have been stained with specific dyes (e.g., ethidium
bromide for DNA) or labeled with fluorescence. The
system uses a specialized camera and illumination
system to visualize and record the gel under different
types of light (e.g., UV, visible, or fluorescence).
2. Fluorescence Detection: Many GelDoc systems are
equipped with UV light sources or other forms of
excitation light, which are necessary for visualizing
stained gels under the appropriate wavelength. For
example, DNA is often stained with ethidium
bromide, which fluoresces under UV light, allowing
the bands to be seen and analyzed.
3. Image Capture and Documentation: The GelDoc
system captures high-resolution digital images of the
gel, which can be saved and analyzed. These images
are crucial for documenting experimental results and
comparing bands between different samples, making
them useful for reproducible results in research and
diagnostics.

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Hybridization oven
A hybridization oven is a laboratory instrument used to incubate samples at a controlled
temperature during hybridization processes, particularly in molecular biology and genomics
experiments. Hybridization refers to the process where
complementary strands of nucleic acids (DNA or RNA) bind
to each other to form double-stranded molecules. The
hybridization oven helps maintain a consistent and optimal
temperature, which is crucial for the success of various
hybridization techniques.

Autoclave

An autoclave is a laboratory device used for sterilizing equipment, media, and other materials by
subjecting them to high-pressure steam at elevated temperatures. The
process of autoclaving is crucial for ensuring that instruments, glassware,
and other items are free from microorganisms, including bacteria, viruses,
fungi, and spores, making them safe for subsequent use in experiments or
clinical settings.

Key Features and Functions:

1. Sterilization Process:
o Heat and Pressure: The autoclave works by using
pressurized steam to achieve sterilization. The typical
sterilization conditions involve heating the material to
temperatures between 121°C to 134°C (250°F to 273°F),
with pressure typically around 15-30 psi (pounds per
square inch). This high temperature and pressure kill
microorganisms, including tough bacterial spores that are
resistant to normal disinfecting methods.
o Duration: The sterilization cycle usually lasts anywhere from 15 minutes to over
an hour, depending on the size of the load and the specific items being sterilized.

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Stainless steel test tube rack

A stainless steel test tube rack is a laboratory tool used to hold test tubes in an organized and
secure manner during experiments. It is made of durable stainless steel, which offers excellent
resistance to corrosion, rust, and staining, making it ideal for use in laboratories where the rack
will be exposed to various chemicals, cleaning agents, and high temperatures.

Key Features and Functions:

1. Material - Stainless Steel:

o Corrosion Resistance: Stainless steel
is resistant to corrosion and rust,
making the test tube rack durable and
long-lasting, even in environments
where exposure to chemicals, water, or
high humidity is frequent.
o Heat Resistance: Stainless steel can
withstand high temperatures, so it can
be used in laboratories where
sterilization or exposure to heat is
necessary (e.g., when test tubes are autoclaved).

Sartorius Arium Bag Tank 50

The Sartorius Arium Bag Tank 50 is a laboratory-grade device used for the storage and
distribution of ultrapure water. It is specifically designed to work in conjunction with the
Sartorius Arium water purification systems, which produce
ultrapure water that meets the stringent requirements of analytical
laboratories, pharmaceutical companies, and other settings where
high-quality water is essential for experiments and production
processes.

Key Features and Functions:

1. Water Storage:
o The Sartorius Arium Bag Tank 50 is a storage system
that holds ultrapure water produced by the Arium water
purification systems. The tank has a 50-liter capacity,
making it suitable for medium-scale laboratory
operations where a consistent and high volume of
ultrapure water is required.
o The tank ensures that the purified water is stored in a
clean, contamination-free environment and is ready for
use at any time. It helps maintain a steady supply of purified water for various
laboratory tasks.

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Ball mill

A ball mill is a type of grinder used to grind and blend materials

for use in various laboratory and industrial applications. It is
commonly used for material processing, grinding solid substances
into fine powder, and mixing materials to achieve a desired
consistency. Ball mills are used in various industries such as
pharmaceuticals, chemicals, ceramics, mining, and construction
materials.

Key Features and Functions:

1. Grinding Process:
o A ball mill consists of a rotating cylindrical
container (the mill shell) filled with steel or
ceramic balls (the grinding media). As the cylinder rotates, the balls inside are
lifted up and then dropped onto the material being ground. The impact and
friction from the balls break down the material into
smaller particles, effectively reducing its size.

Heat block

A heat block (also known as a dry bath incubator) is a laboratory

device used to heat samples to specific temperatures, typically in the
range of 4°C to 150°C (though some models can go higher), for
various applications. It provides a controlled and uniform
temperature environment for sample incubation or heating, and it is
often used when precise temperature control is required without the
use of water baths.

Key Features and Functions:

1. Uniform Heating:
o A heat block provides uniform and stable heating across the entire block, ensuring
that samples (e.g., test tubes, micro centrifuge
tubes, PCR tubes, or vials) are heated evenly.

Sartorius Bottle-Top Dispenser

The Sartorius Bottle-Top Dispenser is a laboratory device used

for dispensing precise amounts of liquids from a large bottle or
container without the need for transferring the liquid to smaller
vessels. It is designed to make the dispensing of reagents,
solvents, acids, bases, or other liquids safer, more efficient, and
more accurate in a laboratory setting.

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Key Features and Functions:

1. Precise Liquid Dispensing:

o The Sartorius Bottle-Top Dispenser allows for precise and accurate dispensing of
liquids in small, controlled volumes. The dispenser typically comes with a dial or
digital display that allows the user to set the volume to be dispensed, ranging from
microliters to milliliters (depending on the model).

Mini centrifuge
A mini centrifuge is a compact and portable laboratory device
used to spin small samples at high speeds in order to separate
components based on their density. It is designed for use with
micro centrifuge tubes (typically 1.5 mL or 2.0 mL) and is
commonly used in laboratories for small-scale sample
preparation, particularly in molecular biology.

Sartorius Arium Comfort Water Purification System

The Sartorius Arium Comfort Water Purification System is a laboratory-grade water

purification system designed to produce ultrapure and purified water from tap or feed water. It is
specifically designed for applications that require high-quality water for a variety of laboratory
processes, such as biological research, cell culture, molecular biology, and analytical procedures.
This system ensures that the water meets the stringent requirements for laboratory work where
water quality is critical.

Key Features and Functions:

1. Water Purification:
o The Sartorius Arium Comfort system purifies
tap water or feed water to produce water of
different grades, including Type 1 (ultrapure)
and Type 2 (pure) water. Type 1 water is
typically used for high-sensitivity
experiments, such as those involving
molecular biology or chromatography, while
Type 2 water is sufficient for applications
like buffer preparation and washing
glassware.

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Thermo Shaker

A Thermo Shaker is a laboratory device used to simultaneously heat and shake samples in
containers such as test tubes, micro centrifuge tubes, or vials. It is commonly used in biological,
chemical, and clinical laboratories for various types of sample incubation, mixing, and reaction
processes that require both temperature control and agitation.

Key Features and Functions:

1. Heating and Shaking:

o The primary function of a thermo shaker is to
provide both controlled heating and mechanical
shaking to samples, making it ideal for
applications where heat and agitation are needed
simultaneously.
o The shaking motion (often orbital shaking)
helps to mix the samples thoroughly, ensuring
even heat distribution and enhancing the speed
and efficiency of chemical or biological
reactions.

Thermo Scientific Spin Adapter

The Thermo Scientific Spin Adapter is an accessory designed to be used with centrifuges,
specifically those that use micro centrifuge tubes or PCR
tubes. It allows users to spin different types of sample
containers, such as 96-well plates, deep-well plates, or larger
tubes, in a centrifuge. The spin adapter facilitates the rotation
of these samples at high speeds, allowing for efficient sample
processing, while maintaining the stability and proper
alignment of the containers during centrifugation.

Key Features and Functions:

1. Adaptation for Multiple Containers:

o The spin adapter is used to convert the
centrifuge rotor into a more versatile platform, enabling it to accommodate
different types of sample containers (e.g., 96-well plates, deep-well plates, or
PCR plates) instead of standard micro centrifuge tubes or test tubes.

Magnetic Heat Stirrer

A Magnetic Heat Stirrer is a laboratory device that combines the functions of heating and
stirring liquids or solutions. It uses a magnetic stir bar to create agitation within the liquid
while simultaneously providing controlled heating. This combination is especially useful for

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processes that require both mixing and temperature control, such as in chemical reactions,
biological assays, or solution preparation.

Key Features and Functions:

1. Magnetic Stirring:
o The device includes a magnetic stir bar (a
small, usually cylindrical or bar-shaped
magnet) that is placed inside the liquid
container.

PCR (Polymerase Chain Reaction) Thermal

Cycler

A PCR (Polymerase Chain Reaction) Thermal Cycler, also known simply as a Thermal
Cycler or PCR Machine, is a laboratory device used to amplify and replicate small segments of
DNA through the polymerase chain reaction process. The PCR process is widely used in
molecular biology for DNA amplification, genetic analysis, diagnostics, and research. The
thermal cycler automates the precise temperature changes required to carry out the PCR process
efficiently.

Key Features and Functions:

1. Temperature Control:
o The PCR thermal cycler is equipped with a highly accurate temperature control
system that can quickly and precisely adjust the temperature of the sample. This
is critical for the multiple steps of the PCR process, which requires different
temperatures for each phase of the reaction.
o The device typically operates with programmable temperature cycles that can
vary between specific ranges (e.g., 50–95°C) to facilitate each stage of PCR.
2. Temperature Stages:
o Denaturation (usually 94–98°C): The DNA sample is heated to a high
temperature to separate the two strands of the DNA double helix.
o Annealing (usually 50–65°C): The reaction temperature is lowered so that short
DNA primers can bind (anneal) to the complementary sequences on the single-
stranded DNA template.
o Extension or Elongation (usually 68–75°C): The temperature is raised to the
optimal range for the DNA polymerase enzyme to synthesize new strands of DNA
by extending the primers along the single-stranded DNA templates.

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 Internship Report: The Helica® Aflatoxin M1 ELISA Kit Laboratory on
Central Laboratory Practices at Hawassa University Agricultural Campus

Introduction

Molecular biology is a fundamental branch of biology that explores the structure and function of
molecules essential for life, such as DNA, RNA, and proteins. Molecular laboratories play a
crucial role in advancing scientific understanding and applications, particularly in biotechnology,
agriculture, and healthcare. At Hawassa University Agricultural Campus, the molecular
laboratory serves as a hub for cutting-edge research and student training, providing insights into
genetic mechanisms and innovative solutions for agricultural and environmental challenges.

This report documents my internship experience at Hawassa University Agricultural Campus,

focusing on the theoretical and practical aspects of molecular laboratory operations. The report
aims to provide a detailed understanding of the components, procedures, and applications
encountered during the internship, with an emphasis on enzyme-linked immunosorbent assay
(ELISA) for aflatoxin detection, as described in the Helica® Aflatoxin M1 ELISA Kit manual.

Objectives

 To gain practical knowledge of molecular laboratory techniques and procedures.

 To understand the principles and applications of ELISA in detecting contaminants like
aflatoxins.
 To develop technical skills in sample preparation, assay execution, and data
interpretation.
 To appreciate the role of molecular tools in ensuring food safety and improving
agricultural outcomes.

Central Laboratory Components The molecular laboratory at Hawassa University Agricultural

Campus is equipped with state-of-the-art tools and resources, including:

1. Centrifuges: Essential for sample preparation and separation of components by density.

2. Micro plate Readers: Used to measure absorbance in ELISA assays.
3. Pipettes and Micropipettes: For precise liquid handling.
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 4. Incubators: To maintain specific temperature conditions for reactions.
5. Glassware and Consumables: Such as test tubes, pipette tips, and microwell plates.
6. Biological Safety Equipment: Including lab coats, gloves, and biosafety cabinets.
7. Reagents and Chemicals: For specific assays, such as antibodies, substrates, and stop
solutions in ELISA.

Procedures in ELISA for Aflatoxin Detection, Preparation of Samples

 Raw Milk: Refrigerate overnight or centrifuge to separate fat, then use the skimmed milk
for analysis.
 Cheese: Grate, extract with methanol, centrifuge, and reconstitute with skim milk for
testing.

Assay Procedure

 The reagents were brought to room temperature before being used.

 The pouch was unsealed, and the required number of wells for the standards and samples to be
tested was removed.
 Unused wells were returned to the pouch, and the pouch was re-sealed to prevent moisture
entry. (The well holder was retained for future use after the assay.)
 Using fresh pipette tips for each, 200 µL aliquots of standards and samples were dispensed into
the appropriate wells in duplicate.
 The plate was covered with sealing tape to prevent evaporation and to protect it from UV light.
 The plate was incubated at ambient temperature (19–25°C) for 2 hours.
 The contents of the wells were discarded into an appropriate receptacle. The wells were washed
by filling them with PBS-Tween 20® from a wash bottle or multi-channel pipette, and the
washings were immediately discarded into a receptacle. This process was repeated three times,
and the wells were tapped face down on absorbent paper to remove residual wash buffer.
 100 µL of conjugate (green cap) was added to each well.
 The plate was re-sealed and incubated at ambient temperature for 15 minutes.
 Step 7 was repeated.
 100 µL of enzyme substrate (TMB) was added to each well, and the plate was incubated for 15
minutes. The plate was covered to protect it from direct light.
 The reaction was stopped by adding 100 µL of stop solution, causing the blue color to change
to yellow.

 The optical density (OD) of each micro well was measured using a micro plate reader at 450
nm with an air blank or a differential filter of 630 nm.

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OBSERVATIONS -

FIG – color changes of ELISA plate FIG – optical

density from micro plate

DATA ANALYSIS USING Data Analysis Worksheet of the

Aflatoxin B1 ELISA Kit (Cat No. 941BAFL01B1-96) (KIT5004).
Aflatoxin B1 is a toxic substance produced by certain molds (fungi) that can contaminate food
like grains, nuts, and spices. This kit is used to detect and measure the amount of Aflatoxin B1
in a sample (like food or animal feed) to ensure it is safe for consumption. The test uses ELISA
(Enzyme-Linked Immunosorbent Assay), a method that involves an antibody specifically reacting with
Aflatoxin B1, allowing its presence and amount to be measured.

Once you have the absorbance values for your samples, you use the standard curve to calculate
their concentration of Aflatoxin B1. This gives you the final result, often in ppb (parts per
billion). Interpretation:

 If the concentration of Aflatoxin B1 in a sample is higher than the safe limit (set by
health authorities), that sample might be unsafe for consumption.

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KEY – OD1 OD2
S1 (Standardized kite 1) = 1.321, 1.342
S2 (Standardized kite 2) = 1.23, 1.209
S3 (Standardized kite 3) = 1.121, 1.081
S4 (Standardized kite 4) = 0.843, 0.854
S5 (Sample A) = 1.013, 0.989
S6 (Sample B) = 0.938, 0.988

The values provided in the image relate to the standard curve used for quantifying Aflatoxin B1
concentrations in a sample. Here's a simple breakdown:

1. R² (R-squared = 0.6037):
o This measures how well the data points fit the regression line.
o A value of 0.6037 means that about 60.37% of the variation in the data can be
explained by the line.
o Ideally, you want an R² closer to 1 for a strong correlation. A value of 0.6037
indicates a moderate fit, but it might need improvement for highly accurate
quantification.
2. Slope (-0.4637):
o The slope represents the rate of change of the dependent variable (Log %B/Bo)
with respect to the independent variable (Log concentration).
o A negative slope means there’s an inverse relationship: as the concentration
increases, the %B/Bo decreases, which is typical in competitive ELISA assays.
3. Intercept (0.5629):
o The intercept is the point where the regression line crosses the y-axis (when the
Log concentration is 0).
o This value helps in predicting the response at a baseline concentration.

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  So, for testing, 100 pg of Aflatoxin M1 was spiked into 1 g of finely grated parmesan
cheese and allowed to remain in contact for one hour at ambient temperature.
Following the extraction procedure as described, the average recovery of Aflatoxin
M1 was 60.37%.

Summary:

 The R² value shows a moderate correlation, suggesting the curve can be used but might
not be highly reliable for precise calculations.
 The slope and intercept define the standard curve, which is crucial for determining
unknown sample concentrations based on their optical density (OD).

Applications and Benefits

1. Food Safety: Ensures dairy products meet safety standards by detecting harmful
aflatoxin levels.
2. Agricultural Improvement: Identifies contamination sources, enabling mitigation
strategies in crop and feed production.
3. Research and Development: Supports the development of new methods to enhance
agricultural productivity and public health.
4. Skill Development: Builds technical proficiency in molecular techniques essential for
careers in biotechnology and related fields.

Conclusion This internship provided a comprehensive understanding of molecular laboratory

practices and their applications in food safety and agricultural biotechnology. The hands-on
experience with ELISA techniques for aflatoxin detection deepened my appreciation for the role
of molecular tools in addressing real-world challenges. The knowledge and skills gained will
serve as a strong foundation for future academic and professional endeavors in biotechnology.

Overview of Molecular Techniques and Protocols from Observations

This document provides a concise overview of three key molecular biology techniques and
protocols: DNA Isolation from Bacteria, and DNA Extraction and Purification from Buccal
Swabs. These methods are integral to molecular genetics, diagnostics, and biotechnology
research.

1. DNA Isolation from Bacteria

Purpose:
This technique extracts DNA from bacterial cells for genetic analysis, sequencing, or cloning.

General Steps:

1. Cell Lysis: The bacterial cells are lysed using enzymes or chemical buffers to release their DNA.
2. Removal of Proteins and Other Contaminants: Proteins are degraded using proteases, and
contaminants are removed by washing or precipitation steps.

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 3. DNA Precipitation: Ethanol or isopropanol is used to precipitate DNA from the solution.
4. DNA Purification: The DNA is washed and re suspended in a suitable buffer.

This method requires careful handling to avoid contamination and ensure high-quality DNA
suitable for downstream applications.

2. DNA Extraction and Purification from Buccal Swabs

Purpose:
This protocol is used to isolate and purify genomic DNA from buccal (cheek) cells collected via
swabs. It is a non-invasive method widely used in clinical and forensic settings.

Detailed Protocol:

1. Sample Preparation:
o Place the buccal swab in a 2 mL micro centrifuge tube.
o Add 500 µL of PBS (phosphate-buffered saline) to the tube.
2. Cell Lysis:
o Add 25 µL of OB Protease Solution and 500 µL of BL Buffer.
o Vortex at maximum speeds for 30 seconds and incubate at 65°C for 10 minutes.
3. Swab Removal:
o Discard the buccal swab after the incubation.
4. DNA Precipitation:
o Add 500 µL of 99% ethanol to the tube.
o Vortex at maximum speed for 20 seconds and centrifuge briefly to collect drops.
5. DNA Binding:
o Insert a HiBind DNA Mini Column into a 2 mL collection tube.
o Transfer 750 µL of the sample to the column and centrifuge at 10,000 × g for 1 minute.
o Discard the filtrate and reuse the collection tube.
6. Repetition:
o Repeat the binding step until the entire sample has been processed.
7. Follow-Up:
o Proceed with subsequent steps (e.g., washing and elution) as detailed in the referenced
protocol for DNA extraction from blood and body fluids.

Applications:
This method is essential for genetic testing, forensic identification, and medical research.

Conclusion

The described methods are fundamental in molecular biology and provide a foundation for
various research and diagnostic applications. Mastery of these techniques ensures the acquisition
of high-quality DNA samples, which are critical for reliable experimental outcomes.

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