CHAPTER ONE

INTRODUCTION

1.0 History and Meaning of SIWES

The Students Industrial Work Experience Scheme (SIWES) is a unit under the Vice-Chancellor’s

Office. It was established in 2016. The Students Industrial Work Experience Scheme (SIWES)

is a skills training programme designed to expose and prepare students of universities and other

tertiary institutions for the Industrial Work situation they are likely to meet after graduation.

The company has been formed by a group of professionals having vivid experience and wide

exposure in Information Technology. People involved here are young qualified Engineering

graduates and qualified business graduate from the renowned universities across the country. The

resource personnel working in the company have been consistently providing reliable support

services and consultancy to a wide variety of corporate houses either in the capacity of executive

or as business partner or consultant. Bottom line of the company philosophy is building a long-

term business partnership with its clients where interpersonal relationship, reliability, assured

quality and target oriented modern technology are the major building blocks. It is a company

where professionals from both technical and functional field group together with an objective of

providing appropriate business solutions. It realizes the importance of functional knowledge and

its impact in developing business solutions. We constantly strive to be a leading technology firm

with profound business and functional knowledge. The key to the company's success is the

maintenance of a close working relationship with the clients through ensuring the best possible

solutions to their needs; to establish and maintain a thorough knowledge and understanding of

client's objective and help them maximize the benefits. We want to establish ourselves as the best

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choice in Computing and Information Technology Services, Consultancy and Development by

offering the full spectrum of services.

1.1 About SIWES: (Student Industrial Work Experience Scheme)

Since the aim of our national policy in education is to build a strong and self- reliant nation, from

the government’s decree No.47 of 8th October, 1971 as amended in1990, which led to the

establishment of Industrial Training Fund (ITF) in 1973/1974 and through the formation of this

body (ITF), in the year 1993/1994 and through the formation of this body (ITF), in the year

1993/1974 SIWES was formed. In Nigeria, the current form of Cooperative Education is known

as the Students Industrial Work Experience Scheme (SIWES).

The Students Industrial Work Experience Scheme (SIWES) is a planned and supervised training

intervention based on stated and specific learning and career objectives and geared towards

developing the occupational competencies of the participants. The aim is make education more

relevance and also to bridge the science-related disciplines in tertiary institutions in Nigeria.

SIWES forms part of the approved minimum academic standards in the institutions, and is a core

academic requirement carrying fifteen (15) credit units. This requirement must be met by all

students in various disciplines before graduation.

1.2 Bodies Involved in SIWES

The main bodies involved in Student Industrial Work Experience Scheme are; The tertiary

institutions and the Federal Government through the Industrial Training Fund (ITF).

Other supervising agencies include:

1. National University Commission (NUC)

2. National Board for Technical Education (NBTE)

3. Industry/Employers (NECA, NACCIMA, MAN, Government Establishments)

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 4. Tertiary Institutions (Universities, Polytechnics, Colleges of Education)

5. Student Trainees (Engineering, Science, Technology, NCE Technical).

The functions of these agencies above are to:

1. Ensure adequate funding of the scheme

2. Establish SIWES and accredit SIWES unit in the approved institutions

3. Formulate policies and guideline for participating bodies and institutions as well as

appointing SIWES coordinators and supporting staff

4. Supervise students at their places of attachment and sign their log book and ITF forms.

5. Vet and process students Log books and forward same to ITF area office

6. Ensure payment of all allowances for the students and supervisors.

1.3 Nature and Scope of SIWES

This is based on the number of weeks or months that student is expected to stay for its

attachment. The minimum duration for SIWES should normally be six months, twenty-four

weeks (24) weeks for University Engineers and Technologist. The cumulative total duration of

attachment over the entire period of the course should preferably be not shorter than 240hrs full

time which will take place during term-time or long vocation.

Induction sessions which is conducted by teaching departments to install the concepts of key

skills (skills for learning, employment and life), work place safety and professional expectations,

legalities and ethics.

1.4 Aims and Objectives of SIWES

The specific objectives of SIWES were summarized by the federal government as follows:

1. To provide students with an opportunity to apply their knowledge in real work and actual

practice.

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 2. To make the transition from school to the world of work easier and to enhance students

contacts for later job placement.

3. Advanced countries, with over 100 years of sustained industrial development and

requisite technical and human infrastructure, have been able to adequately implement

industrial training for their students.

4. They also include providing a structural attachment program with emphasis applications,

management and hands-on experience for students to apply knowledge acquired.

It also aids students to acquire practical skill in other to strengthen their work value

Moreover, it helps them to gain interpersonal and entrepreneurial skills and also installs in

them the right kind of work attitudes and professionalism through interactions with peoples in

the organizations and observations of their future role in the tertiary.

1.5 Benefits of Industrial Training

Experts identified industrial experience as necessity for proper job preparation. This is because

productivity is enhanced by experience graduate or new entrance into the world of work really

needs and early exposure to the value and skills of the industry. Therefore, without appropriate

skills and experiences young graduates are not properly trained on work, norms and role behavior

among others, these components will ensure success at the job place.

Today Information and Communication Technology (ICT) is changing the way many jobs are

performed, thus altering the knowledge and skills required of workers. Consequently, a new level

of competency is required of our students. This cannot be sufficiently met by training facilities in

our education institutions hence, the need for collaborative effort between institutions and

industrial sector.

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The major benefits accruing to students who participate conscientiously in industrial training are

the skills and competencies they acquire. These relevant production skills (RPSs) remain a part

of the recipients of industrial training as lifelong assets which cannot be taken away from them.

This is because the knowledge and skills acquired through training are internalized and become

relevant when required to perform jobs or functions. Several other benefits can accrue to students

who participate in industrial training, attributes such as critical thinking, creativity, initiative,

resourcefulness, leadership, time management, presentation skills and interpersonal skills,

amongst others.

1.6 Brief History of Unimaid Biotechnology Laboratory

The Federal Government of Nigeria established a policy on Biotechnology in April, 2001. In

other to make Nigeria a key player in Biotechnology revolution for the benefit of the country.

This saw the establishment of National Biotechnology Development Agency (NABDA) in

November 2001, under the federal ministry of science and technology, as an institutional frame-

work for implementing the national biotechnology policy.

In other to key into this laudable federal programme, the university of Maiduguri established a

Biotechnology Centre on 8th September, 2003. The Centre signed a memorandum of

understanding with NABDA on Wednesday 23rd July, 2008. With this, the centre serves as the

co-ordination Centre for biotechnology activities in the Norh-East Zone of the country. The

Centre is also one of the designated STEP-B Centres’ of excellence established by the federal

government in collaboration with the World Bank.

1.7 Mandates of The Centre

 To promote co-ordinate and develop biotechnology activities in the North-East region.

 To develop the field of Biotechnology and related research activities in the university

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  Support the development of vast biotechnology research of the university and region at

large.

 Serve as liaison between the various unit of the University on research related to the field

of biotechnology

 Serve as a coordinating centre for the various units of the University and the North-East

region in biotechnology research and development

1.8 Mission of the Centre

To develop the field of biotechnology and related research activities in the University and

support the development of the vast bioresource potentials and need oriented biotechnology

research in the North-East region.

The Centre Approaches This Mission Through:

1. Capacity building: The Centre provides postgraduates and non-degree seeking trainees

with the knowledge and skills they need to accomplish their goals.

2. Research: The Centre focus on issues that impact sustainable development in Agriculture,

Environment and health, with the view of addressing issues of regional and National

significance.

3. Information and Technology Services: The Centre is an information portal for those who

seek the services of affiliated sciences who can design and conduct targeted research and

provide consultation

1.9 Resources of the Centre

The activities of the Centre are support in part by the University and NABDA. Resources are

also obtained through grants as well as fees for services provided.

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1.9.1 Goals and Objective of The Centre

1. To facilitate Research and Development (R&D) in Biotechnology in the University

2. Act as a catalyst for National Development of Biotechnology in the North-East Zone of

Nigeria in collaboration with NABDA

3. To facilitate and establish professional relationship with institution of similar mandate

and research within and outside Nigeria.

4. To act in advisory and consultative capacity on matters relating to biotechnology (R&D)

in the zone

5. To organize conferences, seminars, refresher and specialized courses for the advancement

of biotechnology in the community as consistent with the objectives of the Centre in the

Zone.

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 CHAPTER TWO

2.0 MY TECHNICAL EXPERIENCE AT UNIMAID BIOTECHNOLOGY CENTRE

Unimaid Biotechnology Laboratory is so much important to University of Maiduguri and North-

East entirely, this is because of its contribution towards attendingto the needs of students,
educational services for research purposes, and other schools within and outside Nigeria in
regards to research and recommendation.

Different forms of nucleic acid extraction are carried out in the Centre, using varying sample
ranging from samples from animal, such as brain, liver, kidney, heart etc and from plants
samples such as moringa, onion bulb, neem plant etc.

Culturing of media to be used for extraction of either DNA/RNA/Protein etc are also being
carried at Tissue culture laboratory, which is a section of biotechnology Centre, University of
Maiduguri.

2.1 LABORATORY SECTION OF UNIMAID BIOTECHNOLOGY

In other to conveniently perform this huge but important task, the Centre is divided into two (2)
Laboratories. They are: ‐

 Tissue Culture Laboratory

 Molecular Biology Laboratory

2.2 TISSUE CULTURE LABORATORY

Tissue culture laboratory is a booming laboratory that is of high most important to Unimaid

Biotechnology Centre over years ago. The Laboratory knowledge is so readily available that

anyone with genuine interest can do it. It generates enormous financial returns for the Centre

However,

In other to conveniently perform this huge but important task, the Laboratory is divided into six

(6) units. They are:

 Quarantine area
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  Washing area

 Media preparation area

 Aseptic transfer area

 Culture/grow rooms

 Data collection area

This area is essential for prevention of contamination coming from staffs and other visitors

coming into the laboratory. It is present at the beginning of the lab and as well as at the exit of

the lab. This area is primarily for changing of dresses and shoes. There are separate shoes and lab

coats for lab workers.

WASHINGAREA

This area to is for washing the explants or other plant materials. It’s also for cleaning all beakers,

cylinders and flasks which is use for preparing media or measuring ingredients. This area

consists of large sinks and draining boards. This area also has access to distilled water with space

for drying. Thus, it facilitates the preliminary cleaning to kill the microorganisms present on the

surface.

MEDIA PREPARATION AREA

This area is for preparing culture media recipes and for storage of chemicals and glassware. This

area also has hot plates, pH meters, weighing balances and water baths. The important equipment

needs for media preparation in section of the lab are:

 Magnetic stirrer

 Analytical and top-loading balances

 pH meter

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  Refrigerator with freezer or a separate freezer for storing stock solutions, plant hormones

and other chemicals

 Water purification system along with the space to store the purified water

TRANSFER AREA

In ideal conditions, a separate room should be made next to the media preparation area or next to

the area for the media dispensing unit. However, this area is needed to have complete sterile

conditions in order to transfer explants to media containing culture vessels. So the activities for

this room are:

 Transfer of explants to growth media;

 For sub culturing;

 For multiplication transfers; and

 For transferring multiplied shoots to rooting media.

One of the most important key points for this area is that it always has positive pressure airflow

to make sure that air can leave the room without circulating back in.

DATA COLLECTION AREA

Developing tissue culture protocols for different plant species and varieties involves a lot of trial-

and-error experiments. You will be experimenting with culture media recipes for different

growth stages and also with different growth conditions in the culture/grow room.

2.3 MOLECULAR BIOLOGY LABORATORY

The molecular biology lab, we deal and study biological macromolecules mainly DNA, RNA
and Proteins. Their mechanism of gene replication, mutation and expression. As a result of huge
task perform in this laboratory, it is divided into four (IV) rooms, which include;

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 1. Extraction room
2. Quantification room
3. PCR room
4. Gel Electrophoresis room

2.3.1 Extraction Room

Nucleic acids are the building blocks of life in all living things, from plants and animals to
bacteria andviruses. In research, it’s important to first extract RNA and DNA prior to
downstream processes like quantification, PCR, sequencing,restriction enzyme digestions and
ligations,along with many other applications.Along with determining nucleic acid
concentrations, it’s also important to extractthe nucleicacid of interest which could be either
DNA/RNA/Protein to be work on using downstream applications.

All this is easily carried out in at the extraction section of molecular biology laboratory. This
section is so important that after the said tissue or organ of interested has been harvested, it’s the
second step to perform after homogenizing, it’s the stage where our main sample of interest,
which could be DNA/RNA/Protein would be separated from other particles.

Figure 2.1: Steps involves in DNA extraction

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Reagentsandequipmentat the extraction used in extraction of Nucleic acid.

Red cell lysis buffer

 Sucrose: 109.5 g

 Tris (pH 7.6): 1.58 g

 MgCl2: 476 mg

 Triton-X: 10 ml

 Sodium azide: 200 mg

 Distilled water: up to 1L

Cell lysis buffer

 Tris (pH 8.0) 7.85 g

 Disodium EDTA: 6.68 g

 SDS: 20 g

 Distilled water: up to 1L

Cell lysis buffer with guanidine

 Guanidine isothiocyanate: 50 gm

 SDS 2 gm

 IM Sodium citrate (pH 7.0): 2.5 ml

 2-Mercaptoethanol: 0.7 ml

 Distilled water: up to 100 ml

Buffered phenol

 Phenol 250 g

 Distilled water 40 ml

 Place at 65o C for 1-2 hrs

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  Cool and add 300 mg 8-hydroxyquinoline

 Equilibrate by mixing with equal volume of 1M Tris buffer (pH 8.0)

 Carefully remove the supernatant after allowing phenol to settle down

 Repeat twice equilibration with 1M Tris

 Add 0.4 ml 2-mercaptoethanol

 Add 100 ml of 0.1M Tris buffer (pH 8.0)

 Store at 4o C in a dark bottle

Proteinase K

 Proteinase K: 20 mg

 Distilled water: 1 ml

 Make aliquot of 0.5ml and store at -20oC

 Use 20 μl/extraction

M Ammonium acetate

 Ammonium acetate: 57 g

 Distilled water: up to 100ml

Equipment/Materials at the extraction room include the following

 Centrifuge 5430 Eppendorf

 Sterile 1.5ml microcentrifuge tubes Eppendorf

 Spatula

 Water bath Clifton 99816 (65OC)

 Vortex 12081459 bench mark

 Eppendorf micropipette

 Nanodrop spectrophotometer

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  Automatic pipette tips

 Separation rack

 Mortar and pestle

 Isolation Promega kit

Extraction room has a very huge impact in molecular biology laboratory, as a result, all forms of

extraction raging from DNA, RNA, protein extractionneeded for research purpose, seminar or

conferenceare being carried out in this room, the techniques is used to analyze biological

markers in the proteome and genome.

2.3.2 Quantification Room

A great important section of molecular biology, this section tells you whether your extraction is

successful or not. Nucleic acids are the building blocks of life in all living things, from plants

and animals to bacteria andviruses. In research,it’s important to quantify RNA and DNA prior to

downstream processes like sequencing,restriction enzyme digestions and ligations, PCR and

qPCR along with many other applications.Along with determining nucleic acid

concentrations,it’s also important to calculate the ratio of nucleicacid to protein to ascertain

purity before using the sample in downstream applications, knowing the quantity and what your

nucleic acid entails, will determine the success of your next step, and the quantification room is

where this important task is being carried out.

To conveniently carried out this huge task, the quantification method is divided into fluorometric

and spectrophotometric methods for nucleic acid quantification.

fluorometric is used for applications requiring high sensitivity due to minute available amountsof

nucleic acid; the spectrophotometric methodsis more conventionally used from commonnucleic

acid extraction procedures.

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Best Practices in this room

Care should always be taken to minimize sample contamination and ensure both accurate

andprecise spectrophotometricmeasurements. Some useful tips are presented below:

 Use gloves to protect samples from nuclease or other contamination found on human skin

 Ensure the sample vessel (cuvette, microplate, micro-volume surface) is absolutely clean

 Ensure instrumentation is well maintained

 Use the same buffer type for blanks and samples

 Use replicate samples to reinforce accuracy

2.3.3 Polymerase Chain Reaction Room

It’s an important unit in the molecular biology laboratory, where sample of interest from the

quantification is being transported to this unit of molecular biology. The nucleic acid extracted is

make (amplify) from millions to billions of copies of a specific segment of sample of interest

(DNA/RNA etc), which can then be studied in greater detail.

Repetitive cycles involving template denaturation, primer annealing and theextension of

the annealed primers by DNA polymerase, result in the exponential accumulation of a

specificfragment whose termini are defined by 5’ end of the primers. The primer extension

products synthesized in one cycle can serve as a template in the next. Hence the number of target

samples copies approximately doubles at every cycle. Since its inception, PCR has had an

enormous impact in both basic and diagnostic aspects of molecular biology. Like the PCR itself,

the number of applications has been accumulating exponentially. It is therefore recommended

that relevant scientists and laboratories in developing countries like Nigeria should acquire this

simple and relatively inexpensive, but rather robust technology.

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The Basic Protocol of PCR used in Molecular biology Laboratory

The first step of PCR simply entails mixing thetemplate DNA, two

appropriateoligonucleotideprimers, DNA polymerase, deoxyribonucleosidetriphosphates

(dNTPs) and a buffer. Onceassembled, the mixture is cycled many times,usually 30 times

through temperatures that permitdenaturation, annealing and synthesis. The productis then

displayed on an appropriate gel andexamined for yield and specificity 20. Hence, thebasic PCR

protocol may be outlined as follows:

1. Optimize MgCl2 concentration.

2. Prepare a 10X amplification buffer containingMgCl2 at 10-fold the optimalconcentration.

3. Test the optimised 10X amplification buffer bypreparing a reaction cocktail for a single

PCR. IfPCR is successful, prepare more reactioncocktail for storage at –200C.

4. Add 68μl 15μg/ml template DNA and 9μl ofH2O.

5. Add 1μl 2.5U/μl Taq DNA polymerase.

6. Overlay the reaction mixtures with 100μlmineral oil to prevent evaporation.

7. Heat samples for 90secs at 940C (in a bath oran automated thermal cycler) to denature

theDNA.

8. Incubate at 550C for 2 min to anneal.

9. Incubate the sample at 720C for 3 min toextend.

10. Repeat steps 7 to 9 for another 29 cycles.

11. Electrophorese 10μl from each sample on anagarose non-denaturing polyacrylamide gel.

12. Stain the gels with ethidium bromide.

13. Examine the stained gel.

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Optimization of PCR

Despite its apparent simplicity, the PCR is arelatively complicated and, as yet,

incompletelyunderstood biochemical brew, where constantlychanging kinetic interaction among

the severalcomponents determine the quality of the productsobtained.21 Although the results will

be good inmost cases, there are a number of parameters thatcan be explored, if better results are

required, or ifthe reaction fails altogether. These parameters areexamined below:

Primer Selection

There is no set of rules that will ensure thesynthesis of an effective primer pair. Yet more

thananything else, it is the primers that determine thesuccess or failure of an amplification

reaction.

The Buffer

Changes to the PCR reaction buffer will usuallyaffect the outcome of the amplification. In

particular,the concentration of MgCl2 can have a profoundeffect on the specificity and yield of

amplification.

DNA Polymerase

Initially the PCR used the Klenow fragment ofE. coli DNA polymerase to extend the annealed

primers in a rather tedious procedure. Deoxynucleotide Triphosphates The deoxynucleotide

triphosphates (dATP, dCTP, dGTP and dTTP) are usually present at 50to 200μl each. Higher

concentration may tend topromote misincorporations by the polymerase (thermodynamic

infidelity).

Cycling Parameters

In a typical PCR reaction, the double stranded DNA is denatured by briefly heating the sample to

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90 - 950C, the primers are allowed to anneal to theircomplementary sequence by briefly cooling

to 40 -600C followed by heating to 70 - 750C to extend theannealed primers with the Taq

polymerase.

2.3.4 Gel Electrophoresis Room

In this unit of molecular biology laboratory, a wide variety of mechanically stable experimental

formats such as horizontal/vertical electrophoresis in slab gels or electrophoresis in tubes or

capillaries is being carried out. This unit also facilitates post electrophoretic manipulation

making further experimentation possible such as blotting, electro-elution or MS identification

/finger printing of intact proteins or of proteins digested in gel slices. Since gels used in

biochemistry are chemically rather uncreative, they interact minimally with biomolecules during

electrophoresis allowing separation based on physical rather than chemical differences between

sample components, this unit of the laboratory looks into all this aspect.

Figure 2.2: Gel Sample form Gel Electrophoresis room

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2.4 BIOTECHNOLOGY INSTRUMENT/EQUIPMENT

REFRIGERATOR/FREEZER

Are needed to store reagents and PCR (Polymerase Chain Reaction) products at an average

temperature of 05/-22OC degrees Celsius. PCR is used for the analysis of gene expression,

cloning, sequencing, mutagenesis, and genotyping.

Figure 2.3: Refrigerator/freezer

VORTEX MIXER

Vortex mixers are used for mixing liquid components in tubes such as mixing small vials and for

the resuspension of cells. These mixers are usually designed with touch or continuous modes and

electronic speed control for constant speeds.

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Figure 2.4: Vortex

CENTRIFUGE

The molecular diagnostic lab should also include a refrigerated centrifuge with a minimum of

2000 rpm and non-refrigerated centrifuge depending on the temperature needed. Centrifuges are

used to separate components based on density. Microcentrifuges are also used and should have a

minimum of 14,000 rpm for 1.5ml tubes.

Figure 2.5: Centrifuge

SPECTROPHOTOMETER

Spectrophotometers determine the nucleic acid quantification by measuring light absorption or

the quantity of chemicals in a solution. A light beam passes through the sample and the

compounds transmit light over a wavelength.

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Figure 2.6: Spectrophotometer

DNA SEQUENCER

Sequencers are used to observe the DNA’s molecular sequence. The resulting patterns of DNA

bands or sequences show how enzymes affect the DNA. A DNA sequencer is used to determine

the order of guanine, cytosine, adenine, and thymine which is known as a text string.

Figure 2.7: DNA Sequencer

MICROSCOPE

Microscopes are used for observing and evaluating samples. The microscope should be equipped

with a camera system for archiving test results.

Figure 2.8: Microscope

ELECTROPHORESIS SYSTEM

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An electrophoresis system is used in the analysis of PCR. Molecular movement and separation

can be observed during the electrophoresis process.

Figure 2.9: Electrophoresis System

AUTOCLAVE

Autoclaves are used in molecular diagnostic labs to sterilize waste and lab equipment.

Sterilization is accomplished by using heat to kill bacteria and spores.

Figure 2.9.1: Autoclave

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 CHAPTER THREE

3.0 TECHNICAL REPORT

During the course of my six (6) months industrial training, I worked in the units of molecular

biology laboratory listed below;

 Extraction Room

 Quantification Room

 Polymerase Chain Reaction (PCR) Cabinet

 Gel Electrophoresis Desk

3.1 EXTRACTION ROOM

A method for the extraction of nucleic acids from a wide range of environmental samples was

developed. This method consists of several modules, which can be individually modified to

maximize yields in extractions of DNA and RNA or separations of DNA pools. Extraction of

nucleic acids yields either DNA, RNA or protein, which are higher in quantity and quality with

widely used commercial kits, indicating an advantage to optimizing extraction procedures to

match specific sample characteristics. The ability to separate soluble extracellular DNA pools

without cell lysis from intracellular and particle complexed DNA pools may enable new insights

into the cycling and preservation of DNA in environmental samples in the future. In general the

nucleic acid of interest extracted is subdivided into;

 DNA Extraction

 RNA Extraction

3.1.1 DNA Extraction

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The first step in DNA analysis is to get a good quality sample; a process commonly known as

“extraction”. Since DNA is a very long molecule it can easily get broken by vigorous shaking

during the process of extraction. Therefore, gentle and careful handling in the processing is

essential. DNA can also be destroyed by DNase that is commonly present in the environment or

in the bacteria that may contaminate the sample.

There are three basic steps in a DNA extraction:

1. Removing the membrane lipids by detergents to expose DNA in the nucleus of the cell.

2. Removal of proteins by protease digestion and subsequent precipitation by phenol or

other agents.

3. Precipitation of DNA with ethanol or isopropanol.

Sources of DNA

DNA can be extracted from any source that contains nucleated cells. It is most commonly

extracted from blood collected in EDTA. The blood may be kept at 4 OC for a few days without

causing any significant loss in the yield of DNA. DNA can also be extracted from bone marrow

aspirates or bone marrow smears on slides. Archival bone marrow slides stored at room

temperature for several years have been used to extract good quality DNA. Buccal smear on

cotton swab or mouth wash is another easily available source of DNA. This is especially useful

for field work. Solid fresh tissues, like surgical biopsy specimens, chorionic villi and tissues

collected at autopsy are also used for DNA extraction. DNA can also be extracted from hair root,

blood stains, archival bones etc. Fixation of the tissue with formaline can make DNA extraction

very difficult. Special processing protocols may be required to extract DNA from paraffin

embedded tissues.

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Figure 3.1: Rat Kidney for DNA Extraction

Choice of the method

The standard method of DNA extraction uses phenol chloroform for protein precipitation.

Keeping in view the toxicity of phenol, methods have been developed to precipitate proteins

without using phenol. A large number of commercial kits are also available that are time as well

as cost effective. Some of the methods can also be automated for large Extraction of Nucleic

Acids scale DNA extraction. A quick method of DNA extraction is by ion exchange resin Chelex

100.

In the subsequent unit phenol chloroform method is described in detail. It is robust and cost

effective and consistently gives good quality high molecular weight DNA.

DNA extraction from CVS and fresh tissues

1. Take approximately 25-50mg of fresh tissue (chorionic villi, skin, or other solid tissues)

in 0.5ml of cell lysis buffer (Table 2.1).

2. Add 20-40μl of Proteinase-K depending on the amount of tissue.

3. Keep at 37oC overnight. Allow longer incubation or add more Proteinase-K if the tissue

is not completely digested/dissolved.

4. Proceed as step 11 onwards of the DNA extraction protocol.

DNA extraction from archival bone marrow slides

1. Take a slide of bone marrow smear that has good number of cells.

2. Layer about 0.7ml cell lysis buffer (Table 2.1) on the smear.

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 3. Gently scratch the smear from the slide with a wooden stick and transfer the contents to

an Eppendorf tube.

4. Add 20μl Proteinase-K (Table 2.1) and keep at 37oC overnight.

5. Cell lysis buffer with guanidine (Table 2.1) may be used instead of the standard cell lysis

buffer and Proteinase-K.

6. Proceed as step 11 onwards of the DNA extraction protocol.

Chelex method of DNA extraction

1. Make 5-7% solution of chelex, aliquot 300μl in 1.5ml Eppendorf tubes and refrigerate.

2. Take 300μl blood and add 3ml distilled water or RBC lysing solution to lyse the red cells.

3. Centrifuge at 5000 rpm for 2 minutes to pallet the white cells.

4. Repeat red cell lysis step if the white cell pallet contains too many red cells.

5. Add 300μl 5-7% chelex solution to the white cell pallet and vortex for 15-20 seconds.

6. Place the tube in a heating block at 95oC for 20 minutes.

7. Vortex for 15-20 seconds.

8. Centrifuge at 10,000 rpm for 2 minutes.

9. Transfer the supernatant to a fresh Eppendorf tube and use as source of DNA.

10. The DNA extracted by Chelex method may contain some residual haemoglobin

especially when the white cell pallet contains red cells. Such DNA may give excessive

background fluorescence in real time PCR applications.

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Figure 3.2: During DNA Extraction

3.2 QUANTIFICATION OR MEASUREMENT ROOM

Most PCR applications work well at DNA concentration of 100-300ng/μl. This concentration can

be achieved by following the guidelines given in the extraction protocol. However, in

applications using genetic analyzer it becomes very critical to know the exact concentration of

DNA. In quantification room, there are several ways or methods available to know the DNA

concentration and its purity.

Optical density (OD) method

DNA and RNA absorb UV light at 260nm. The OD of DNA solution measured at 260nm can be

used to calculate the concentration of DNA or RNA. The following example can be used to

calculate DNA in an unknown solution:

 Make 1: 100 dilution of DNA in distilled water (20μl + 2ml)

 Take OD at 260nm

 DNA concentration (ng/μl) = 50 x dilution factor x OD

Example:

 OD at 260nm: 0.068

 Concentration: 50 x 100 x 0.068 = 340 ng/μl or 0.340 μg/ml

27
Figure 3.3: Quantifying DNA sample using Nanodrop

The optical density method may also be used to determine the protein content of DNA. Proteins

left over from the extraction procedure can interfere in PCR and therefore it sometimes is

required to know the purity of the extracted DNA. Proteins absorb UV light at 280nm. In a good

DNA sample the ratio of OD at 260nm and 280nm should be above 1.8. Ratio below 1.8

indicates protein contamination in the DNA solution.

Fluorometry Method

Commercial kits based on fluorescent dyes like SYBR Green can be used for DNA

quantification. The fluorescence given by standards of known DNA concentration is used to

know the concentration of an unknown sample of DNA.

Figure 3.4: Measuring RNA quantity and Quality Using Nanodrop

3.3 POLYMERASE CHAIN REACTION ROOM

Having worked in a molecular biology laboratory as a SIWES student I was able to lay my hand

on polymerase chain reaction (PCR). PCR is an in vitro method in which a small amount of

DNA can be copied many times in a short time period. PCR was invented in the early 1980s by

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Kary B. Mullis who later shared a Nobel Prize in Chemistry for his work. Since then, PCR has

become a standard and essential practice in molecular biology and can be used in a multitude of

scientific techniques such as molecular cloning or molecular diagnostics.

Steps involves in carrying out PCR

The beauty of PCR is that it can amplify DNA using only a short list of reagents and several

heating and cooling steps. PCR relies on heat resistant DNA polymerase from the thermophilic

bacterium, Thermosaquaticus (Taq). Taq polymerase is thus a heat resistant enzyme that can

withstand changes in temperature. Taq was first identified in the late 1960s during research at hot

springs in yellow stone National Park. In addition to Taq DNA polymerase, PCR requires free

nucleotides (dNTPs), template DNA to amplify from and unique single stranded DNA primers

that bind upstream (5’) and downstream (3’) of the DNA region of interest. Primers are crucial

for this process as DNA polymerases require an existing strand of DNA to add nucleotides to.

Using these reagents and a series of heating (denaturing) and cooling (annealing) steps Taq

polymerase can copy DNA between the primers using the dNTPs.

Below are the 3 specifics basic steps of a PCR reaction:

 Denaturation

To amplify DNA, the two strands of the template DNA first have to be separated. This occurs by

heating the dsDNA template to a point where the hydrogen bonds break between the base pairs.

This results in the separation of the two DNA strands.

 Annealing

The temperature is then dropped to a range in which the forward and reverse primers are stable.

At this temperature the primers can anneal to the single stranded DNA template strands. DNA

polymerase is also stable at this temperature and can bind to the primers.

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  Extension

The temperature is then raised slightly to Taq polymerase’s ideal temperature (70-75 OC). At this

temperature Taq polymerase can synthesize and elongate the target DNA quickly and accurately.

Figure 3.5: Step involves of PCR in thermocycler

Types of PCR

Since the invention of PCR, different PCR methods have been developed for different scientific

applications. These PCR methods all use the same basic PCR set up and steps but differ in how

the PCR products are analyzed.

End point PCR

End point PCR, as the name implies, analyzes the end product of PCR temperature cycling. The

final PCR product is often visualized on a diagnostic agarose gel to confirm product presence,

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size, and relative quantity. End point PCR is most commonly used in molecular cloning,

sequencing, and genotyping. It’s extremely useful but is not as quantitative as other methods of

PCR. Theoretically scientists should be able to determine the quantity of DNA after a PCR

reaction as the amplicon doubles every reaction cycle. However, it is common for dNTPs and

other reagents to run low during the final cycles which would slow or stop PCR amplification.

Quantitative polymerase chain reaction, qPCR

Quantitative polymerase chain reaction (qPCR) also known as real time PCR is a PCR technique

used for measuring a starting DNA concentration using PCR. qPCR requires the addition of a

probe based fluorescent dye that intercalates with any dsDNA and the use of a fluorometer

feature built into the thermocycler to measure that fluorescent output. With this fluorescent dye,

the concentration of the DNA during the PCR reaction cycles is continuously detected via a

fluorescent signal. The signal increases proportionally to the amount of product produced each

cycle.

To determine the concentration of the starting template DNA the fluorescent signal throughout

the reaction is compared to a standard curve of amplified DNA of a known starting

concentration. The cycle in which the unknown DNA is detected compared to the standard curve

can be used to determine the amount of starting material in your sample.

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Figure 3.6: Real time and Conventional PCR Machine

3.5 GEL ELECTROPHORESIS ROOM

Gel electrophoresis is a widely used technique in life science laboratories to separate

macromolecules such as DNA, RNA, and proteins. In this technique, molecules are separate

based on their size and electric charge. Gel electrophoresis is usually performed in labs to

analyze DNA, RNA, or protein samples from various sources.

Principles of gel electrophoresis

The gel electrophoresis technique exploits the difference in size and charge of different

molecules in a sample. The DNA or protein sample to be separated is loaded on to a porous gel

placed in an ionic buffer medium. On application of electric charge, each molecule having

different size and charge will move through the gel at different speeds. The porous gel used in

this technique acts as a molecular sieve that separates bigger molecules from the smaller ones.

Smaller molecules move faster across the gel while the bulkier ones are left behind. The mobility

of the particles is also controlled by their individual electric charge. Two oppositely charged

electrodes that are part of the system pull molecules of towards them on the basis of their charge.

How Gel Electrophoresis Work

The gel used in gel electrophoresis is usually made of a material called agarose, which is a

gelatinous substance extracted from seaweed. This porous gel could be used to separate

macromolecules of many different sizes. The gel is submerged in a salt buffer solution in an

electrophoresis chamber. Tris-borate- EDTA (TBE) is commonly used as the buffer. Its main

function is to control the pH of the system. The chamber has two electrodes – one positive and

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another negative - at its two ends. Samples that need to be analyzed are then loaded into tiny

wells in the gel with the help of a pipette. Once loading is complete, an electrical current of 50-

150 V is applied. Now, charged molecules present in the sample start migrating through the gel

towards the electrodes. Negatively charged molecules move towards the positive electrode and

positively charged molecules migrate towards the negative electrode.

The speed at which each molecule travels through the gel is called its electrophoretic mobility

and is determined mainly by its net charge and size. Strongly charged molecules move faster

than weakly charged ones. Smaller molecules run faster leaving behind the larger ones. Thus,

strong charge and small size increases a molecule’s electrophoretic mobility, while weak charge

and large size decreases the mobility of a molecule. When all molecules in a sample are of the

same size, the separation will solely be based on their size. Once the separation is complete, the

gel is stained with a dye to reveal the separation bands. Ethidium bromide is a fluorescent dye

commonly used in gel electrophoresis. The gel is soaked in a diluted ethidium bromide solution

and then placed on a UV transilluminator to visualize the separation bands. The bands are

immediately examined or photographed for future reference, as they will diffuse into the gel over

time. The dye can also be loaded into the gel well in advance to track the migration of the

molecules as it happens.

Applications of gel electrophoresis

Gel electrophoresis is widely used in the molecular biology and biochemistry labs in areas such

as forensic science, conservational biology, and medicine.

 Some key applications of the technique are listed below:

 In the separation of DNA fragments for DNA fingerprinting to investigate crime scenes

 To analyze results of polymerase chain reaction

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  To analyze genes associated with a particular illness

 In DNA profiling for taxonomy studies to distinguish different species

 In paternity testing using DNA fingerprinting

 In the study of structure and function of proteins

 In the analysis of antibiotic resistance

 In blotting techniques for analysis of macromolecules

 In the study of evolutionary relationships by analyzing genetic similarity among

populations or species.

GELL IMAGEHJG K HIOLUUUUUUUUUUUUUUUUUHIIHKKKKKKKKKKKK

Figure 3.7: Loading of Gel with DNA sample

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 CHAPTER FOUR

4.0 SUMMARY, CONCLUSION AND RECOMMENDATION

4.1 SUMMARY

SIWES Programme is bridging the gap between classroom and the work-place by giving

students the opportunity to apply the knowledge and skills they have learned in school to real

world situation. There are number of benefits to participating in SIWES, including Improved

perspectives, enhanced skills, better understanding of the industry, increases self confidence in

students among others. Overall, SIWES is valuable program for students in technical and

vocational fields, as it provides them with practical experience that can help them succeed in

their careers.

4.1.1 ADVICE FOR THE COMING PARTICIPANTS

 Students should always be attentive, obedient and respect each other especially their

tutors.

 Dedication of maximum time and resources.

 Establishment of self-confidence to make learning easier.

 Watching tutorials on what they have been taught fills the missing gaps.

4.1.2 ADVICE FOR THE SIWES MANAGERS

 Supervisors should always visit students monthly in their various places of attachment.

 Managers should be very supportive and forgiving to students with genuine reasons.

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  Allowances should be paid to students during their program just like NYSC and not after.

This would help them a lot on how to manage financial problems during their training

course.

 Managers should provide a well facilitated place of attachment for students.

 Place of attachment should be considered according to student`s residential area.

4.2 CONCLUSION

 My six (6) months Industrial Training at Unimaid Biotechnology Centre was a huge

success and a great time of acquisition of knowledge and skills. Through my training I

was able to appreciate my chosen course of study even more, because I had the

opportunity to blend the theoretical knowledge acquired from school with the practical

hands-on application of knowledge gained here to perform very important tasks that

contributed in a way to my productivity in the company. My training here has given me a

broader view to the importance and relevance of biochemistry in the immediate society

and the world as a whole, as I now look forward to impacting it positively after

graduation. I have also been able to improve my communication and presentation skills

and thereby developed good relationship with my fellow colleagues at work. I have also

been able to appreciate the connection between my course of study and other disciplines

in producing a successful result.

4.3 RECOMMENDATION

There are several ways the SIWES program could be improved:

 Improve the matching process: Ensuring that students are placed in companies or

organizations that are relevant to their fields of study and that aligns with their career goals

can help improve the overall effectiveness of the program.

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 Offer more support to students: Providing students with more supports during their work

placement, such as through mentors or additional training, can help them to get most out of

their experience.

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