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CHAPTER ONE

1.1 BACKGROUND OF SIWES

The students industrial work experience scheme (SIWES) was

established by industrial training fund (ITF) in 1973 to solve the problems

of lack of adequate practical skills preparatory for employment by Nigerian

graduates of tertiary institutions.

It was designed to give Nigerian students studying occupational related

courses in the various institutions the experience that would supplement

their theoretical learning.

The scheme exposes students to industrial based skill necessary for a

smooth transition from a classroom to the world of practical. The scheme is

also aimed at bridging the gap between students existing theoretical

knowledge and practical field experience through attachment to industrial

establishment.

The scheme affords students of tertiary institution the opportunity to

get familiarized and exposed to the needed experience in handling

machinery and equipment which are not usually available in various

institutions. The Student Industrial Work Experience Scheme (SIWES) is

the accepted skill training programs which form part of the approved

minimum academic standards in the various degrees program for all

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Nigerian universities in accordance with the education of Nigeria. It is

founded by federal government of Nigeria and jointly coordinated by

Industrial Training Fund (ITF) and National University Commission (NUC).

1.2 AIMS AND OBJECTIVES OF SIWES

To prepare students for the work situation they are likely to meet

after school.

To afford students an opportunity to develop entrepreneurial skills

and knowledge.

To create partnership between the university and both the private

and public sector enterprises.

To seek industrial evaluation in our degree program in terms of

relevance to industry and commerce.

To give students an opportunity to test their theoretical tools and

connect what they learnt during their first three/four years of

program with the reality of running organization related to their

courses.

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1.3 BRIEF HISTORY OF NATIONAL ROOT CROP RESEARCH

INSTITUTE (NRCRI)

The national root crop research institute (NRCRI) umudike started as

a 20 hectare provincial farm on the 1st January, 1923 under the Nigeria

department of agriculture with headquarters of moor plantation Ibadan in

1955, while the country was recognized, It came under the eastern regional

director of agriculture with headquarters at Enugu, the school of

Agriculture was established close to it in 1955, it became known as the

eastern Nigeria agriculture research station in 1956 but this name changed

to agricultural research and training station (ARTS) in 1965 when the

research and the school of agriculture now Michael Okpara University of

Agriculture(MOUA) where merged.

The federal government of Nigeria took over the station on 1st April

1972 and renamed it Federal Research and Training Station (FARTS).

In 1975, the station was upgraded to a commodity research institute

under decree No. 33 of the federal military government of Nigeria and it

was renamed national root crop research institute (NRCRI) on 1st April

1976 by virtue of decree No. 5 in January 1977, the institute came under

the national science and technology development agency (NSTD).

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In 1980, the institute was transferred to the ministry of science and

technology bill of 1980.

Since 1996, it has been under the federal ministry of agriculture and

rural development (FMARD).

Today, the institute land area is 416 hectare at the headquarter and

1000 hectares at the out station. NRCRI is one of the 18 agricultural

research institute under federal ministry of Agriculture and rural

developmental and the only agro-based research institute east of Nigeria.

1.4 THE OUTSTATIONS

The Institute outstations are 6 in number and cover 3 geopolitical zones as

follows:

Jos Outstation, Kuru - North Central

Otobi – Benue Stations - North Central

Igbariam – Anambra State - South East

Nyanya FCT, Abuja - FCT

Gassol – Taraba State - North East

Kachia, Kaduna State - North Central

1.5 RESEARCH AND INFORMATION

Organization

The Institute is governed by a board whose members are appointed by the

President of the Federal Republic of Nigeria. The daily affairs are run by the

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Director/ Chief Executive officer, assisted by Assistant Directors and Heads

of Division who constitutes the Institute’s Management team. To ensure

efficient and effective delivery of services, the Institute is structured into

divisions, namely; Root Crops Research (RCR). Tuber Crops Research (TCR,

Planning, Monitoring and Evaluation (PME). Research support Services

(RSS). Farming Systems Research and Extension (FSRE), Information and

Documentation (I&D), Administration, Finance and Accounts and Estate

Management. The divisions are subdivided into programs, as follows:

Root Crops Research: Cassava, Sweet potato, and other Root Crops.

Tuber Crops Research: yam Potato, Ginger and Cocoyam.

Farming Systems Research and Extension: Farming Systems

Research and extension services.

Planning, monitoring and Evaluation: Planning, monitoring and

budgeting, Statistics, human Resources Development and Sub-

stations.

Research Support Services: Biotechnology, Computer/meteorology,

Irrigation, biochemistry, Soil and Water Management, Post

Harvest/Garri Processing/WIA, Genetic Resources, Plant Protection,

Plant Breeding and Research Engineering.

Information and Documentation: library, Information and

Documentation

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Finance and Accounts: Accounts: Accounts and Stores.

Administration: Registry, Purchasing, pensions.

1.6 MANDATE OF NATIONAL ROOT CROP RESEARCH INSTITUTE

(NRCRI) UMUDIKE

The national root crop research institute umudike has the national

mandate of conducting researches into the genetic improvement of Root

and Tuber crops namely, yam, cassava, potato, sweet potato, cocoyam,

Hausa potato (Solenostenum esculentus), (Plectranthus esculentus) Tumeric

(Curcuma longa) sugar beet and Radish, as well as research into their

production, storage, processing utilization and marketing etc.

1.7 AIMS OF NATIONAL ROOT CROP RESEARCH INSTITUTE (NRCRI)

UMUDIKE

The national root crop research institute (NRCRI) conducts researches on

cassava, sweet potato, cocoyam, yam and other root crop of economic

importance. Their aims are:

Improvement of the utilization of bi-products.

The improvement of agronomic and husbandry of crops.

The mechanization and improvement of the method of cultivation,

processing and storage of crops.

Multiplication of root and tuber crops through micro-propagation.

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Ecology of pest and diseases of crops and improve method of control

through bulky rate and characterization.

1.8 DEPARTMENT I WAS INVOLVED IN

Biotechnology Department: This department is divided into three

sections:

Plant tissue culture laboratory

Molecular laboratory

Trait profile laboratory

CHAPTER TWO

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2.1 PLANT TISSUE CULTURE LABORATORY

Plant tissue culture is a method of biological research in which

fragments of tissue from plants are introduced into artificial environment

in which they continue to grow and function. Plant tissue culture is a

technique used for in vitro propagation of plants in a controlled artificial

environment, under aseptic conditions. The technique is often used to

produce the clone of a plant. Plant tissue culture employs this newly

developed technology to propagate plants in an advantageous way. It holds

some benefits like quick production of clone plants, production of mature

plants and regeneration of plants in the absence of seeds and pollination

process.

The practice of plant tissue culture has changed the numerous approaches

towards plant propagations. The application of this technology to the

propagation of plants involves micro propagation, which allows the

production of large number of uniform and disease free plants from small

pieces of the stock plant in relatively short period of time depending on the

species in question, the original tissue pieces may be taken from short tip

leaf, lateral buds, stems or root tissues. In most cases, the original plant is

not destroyed in the process.

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The main factor of considerable importance is that once a part of the

plant (explants) is placed on nutrient medium, proliferation of lateral buds

and adventitious shoot or differentiation of shoots directly from callus,

results in tremendous increase in number of shoots. Research institutes

like national root crop research institute (NRCRI) umudike have

established tissue culture facilitated with commercial scale operations

already attained in the mass propagation of root and tuber crops like

cassava, yam, cocoyam, turmeric, potatoes and so on.

2.2 THE BASIC FACILITIES OF PLANT TISSUE CULTURE LABORATORY

They include the following:

Ante-Room: This is a small room sited at the entrance of the

laboratory. This room prevents the inflow of air into the laboratory

to avoid contaminations from natural environment. Also it is where

staffs wear their laboratory coats and change their foot wears to

reduce the introduction of pathogens into the laboratory.

Kitchen/Media Preparatory Room: An area is required for

preparation of media, hence, the media preparation room or kitchen.

It provides a space or workbench for media preparation and

dispensing. In this room, provision is also made for placing hot

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plates/stirrer, pH meter, weighing balance, water-bath, oven,

refrigerator, dispensers, distillers etc.

Autoclave Room: As the name implies, this is a room where

autoclaves are fixed for sterilization of media and materials.

Aseptic Manipulation Room: This is where initiation, sub culturing,

surface sterilization is done under the lamina air flow hood. The

temperature of this room is 250c.

Culture Room: This can also be called incubation room. It is a room

for growth promotion and maintenance of plants. This room gives the

initiated explants the adequate conditions required. The culture

room has shelves for storage of plantlets. Each shelf contains

florescent bulbs, which serves as a source of light to the plantlets for

photosynthesis. The light period of photosynthesis last for 16hrs

while dark period last for 8hrs. The temperature of culture room is

280c+ or -2.

Washing Spot: This is a separate area attached to the laboratory

where washing of culture vessels and apparatus takes place.

Screen House: This is a special house with a special roof system

which reduces direct sunshine from getting to the plants. It is where

plantlets are introduced during weaning for gradual adaptation to

natural environment.

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2.3 INSTRUMENTATION

Double Distiller: This instrument is made up of two compartments

each with a condenser. The first compartment has two heating coils

which boil water at red heat. The steam from this compartment

condenses in the second compartment which is also fitted with tap

for collection of distilled water.

Sterilization Oven: This equipment is used to dry and sterilize glass

wares; glass coverlids, glass slides, instruments, pipettes, test-tube

etc at about 1600c per hour. This is to maintain sterility and avoid

contamination.

Hot Box Oven: It is used to dry various samples and baking in some

cases at regulated temperature. For example it is used to dry leaves

at 1600c-1800c for 1-3hrs in order to obtain the dry (weight) mass of

the leaves.

Lamina Air Flow/Hood: It has a small motor to blow air which first

passes through a coarse filter where it losses large particles and

subsequently through a fine filter, the latter known as, The High

Efficiency Particulate Air (HEPA) filter which removes particles

larger than 0.3µm, and the ultra clean air (free from fungal and

bacterial contaminant) flow in a horizontal movement and prevents

air from flowing into the cabinet from outside during aseptic

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manipulation. The hood has a short-wave UV light that can be turned

on for a few minutes to sterilize the surfaces of the hood but only

accessible to the exposed surfaces or area. This lamina air flow is

used to carry out aseptic manipulation such as initiation of explants,

surface sterilization of explants and sub-culturing of plantlets.

Autoclave: this is equipment used for steam sterilization of

apparatus and media at 1210c, 1.05kg/cm2 for 15minutes. Autoclave

achieves sterilization by generating steam under pressure from

water. The steam destroys existing microorganisms when it comes in

contact with them. The higher the temperature the lower the time

required for sterilization.

Precision Dispenser: This equipment has a digital system which

ensures accurate measurement of prepared medium into various

culture vessels. It is used for dispensing accurate amount of a

prepared medium into culture vessels thereby providing uniform

volume of measurement.

pH Meter: This is an instrument used to measure the degree of

concentration of hydrogen and hydroxyl ions of a medium. The

electrode part of the pH meter is stored in potassium chloride

solution (KCL) to ensure sensitivity and accuracy of the instrument. It

works with room temperature at the moment. The pH meter

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temperature scale is adjusted to room temperature using a knob. The

electrode is dipped in the media and 0.5N hydrochloric acid or 0.5N

sodium hydroxide is used to adjust the pH of a medium to 5.8.

Magnetic stirrer: This equipment is made up of electromagnet

which rotates under the influence of electric charge

(electromagnetism). The rotation of the electromagnet causes the

magnetic stirrer to move in response to it. This movement brings

about the stirring effect. It has a heating coil fitted into it which

produces the heating effect as a result of electric charge. This

equipment is used for heating and homogenously mixing of

components of a medium simultaneously.

Analytical Weighing Balance: This equipment is an electrical device

that is used to weigh minute quantity of media components. It has a

very high sensitivity with its degree of accuracy up to four decimal

places. It has to be switched on for one hour prior to usage for

stabilization.

Vacuum Oven: This is used for dry heating of materials under a

temperature of 180-2000c and allowed to heat for 1-3hrs.

Drying Cabinet: This equipment is used for drying apparatus at low

temperature of about 400c.

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Microwave Oven: This equipment is used to melt agar during media

preparation.

Water Bath: This equipment is used for indirect heating of media to

avoid mechanical loss.

Electric Shaker: This is an electrical device that is used for shaking

of solvents with solutes.

Refrigerators: these are used for storage of hormones and media.

Canisters: These are used for storage of Petri dishes for sterilization.

Measuring Cylinders: These glass wares are used for volumetric

measurement of liquids in mls (milliliters).

Culture Vessels: These include beakers, conical flask, Petri dishes,

round and flat bottom flask, test tubes etc. they are used for culture

media preparations.

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2.3 STAGES OF ACTIVITIES IN THE PLANT TISSUE CULTURE

LABORATORY

STAGE I: Preparation of Suitable Nutrient Medium

Culture medium/Growth medium is a solution freed of all

microorganisms by sterilization (usually in an autoclave, where it

undergoes heating under pressure for a specific time) and containing the

substances required for the growth of plants. The medium may be

solidified by the addition of Agar. While some media consist of complex

ingredients such as the plant growth hormone; BAP (Benzene Amino

Purine), NAA (Naphthalene Acetic Acid) etc.

Aim: to prepare an artificial condition of which plant can grow in famine or

food crisis.

Materials/Equipments/Apparatus: media components, 1litre of distilled

water, Magnetic stirrer hot plate, test tubes, analytical weighing balance,

autoclave, pH meter, precision dispenser, magnetic stirrer/rod, culture

vessels etc.

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Procedure in Preparation of 1litre

Double distilled water was poured in a beaker, and magnetic

stirrer/rod was placed in it, after which the beaker was placed on the

magnetic stirrer hot plate for stirring.

The following components were added to the beaker of distilled

water in the required quantity as follows:

Components g or ml/litre

Sucrose 30g

Myo-Inositol 0.1g

Stock I solution 50ml

Stock II solution 5ml

Stock III solution 5ml

Vitamin mixture 5ml

The above components were allowed to dissolve by heating and

stirring to obtain a homogenous mixture. The medium was made up

to the required volume (1000mls).

Since the pH of a medium is 5.8, the pH of the medium was checked

and standardized using buffer indicators of pH 4 and pH 7. If the pH

of the medium is below 5.8 (acidic),NaOH is used to adjust it up to

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5.8, but if the pH is above 5.8(alkaline), Hcl is used to bring the pH

down to 5.8. The significance of adjusting the pH is to ensure

solubility and availability of nutrients and gellability of the gelling

agent.

After the pH of the medium was checked and standardized, 7g of

agar was weighed and added into the medium, after which the

medium was allowed to heat/stir until it becomes clear.

The medium was dispensed in a test tube at a defined volume as

directed with the use of precision dispenser.

The medium was then sterilized with autoclave at a temperature of

1210c, 1.05kg/cm3 for 15minutes.

Precautions

The amount of medium was known

It was ensured that there were enough culture vessels that can

contain the prepared medium.

Composition of 1litre of Stock I Solution

Components g/l

Distilled water 1000mls

Ammonium Nitrate (NH4.NO3) 33g/l

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Potassium Nitrate (KN2) 38g/l

Calcium Chloride Dihydrate (CaCl.2H2O) 880g/l

Magnesium Sulphate Hepahydrate (mgSO4.7H2O) 7.4g/l

Potassium Phosphate (KH2PO4) 3.4g/l

Composition of 1litre of Stock II Solution

Components g/l

Potassium Iodine (KI) 0.16g/l

Boric Acid (H3BO3) 1.26g/l

Zinc Sulphate Heptahydrate (ZnSO4.7H2O) 1.72g/l

Manganese Sulphate Heptahydrate (MnSO4.7H2O) 4.46g/l

Copper Sulphate Pentahydrate (CuSO4.5H20) 0.005g/l

Cobalt Chloride Hexahydrate (COCL2.6H2O) 0.005g/l

Composition of 1litre of Stock III Solution

FeSO4.7H2O 5.560mg/l

Na. EDTA. 2H2O 7.470mg/l

Composition of 1litre of Vitamin Stock Solution

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Thiamine hydrochloride 10mg/l

Pyridoxine 50mg/l

Nicotinic acid 50mg/l

Glycine 100mg/l

STAGE II: Initiation of Explants

This is the establishment of the plant tissue in vitro by sterilizing the

explants and initiating it into culture. Explants are excised from a healthy

mother plant, it was surface sterilized usually with 2.5% sodium

hypochloride (NaOCL) and 70% ethanol finally with TWEEN 20 or

commercial bleach (TWEEN20 is a surfactant, which are wetting agent, it

enhances the penetration of the sterilant into explants for initiation) and

then rinsed severally before initiating into the medium.

It is also necessary to examine the mother plant before using its

tissue. If the mother plant has a disease, then it may transfer it to the clone

explants.

There are types of culture method:

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Nodal culture method: after sterilization of the explants, it was

excised into nodal segment, each nodes having meristematic part

after which you inoculate into the medium.

Meristem tip culture method: in this method, microscope is used to

view so to collect the meristem and then inoculate into the medium.

Aim: The essence of this is to produce many explants and plantlets from

one mother plant.

Materials/Equipments/Apparatus: mother plant, sterilized distilled

water, beakers, Petri dishes, forcep, surgical blade, lamina air flow, scalpel

etc.

Procedure:

The mother plant was washed very well under running tap before

planting in a bucket of sterilized sand at the screen house or net

tunnel and allowed to grow.

The sprouted part of the mother plant was excised with surgical

blade

The explants were washed with detergent and rewashed with

running water severally so to reduce and wash-off some ecto-

pathogens (microbes).

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The explants were soaked in fungicides for 20-30minutes before

decanting.

Note: All the equipments/apparatus to be used were sterilized in the

autoclave for 30minutes at a temperature of 1210c, 1.05kg/cm3 and

allowed to cool.

The explants were put into 70% ethanol in a culture vessel and

covered; it was then placed on electronic shaker to shake vigorously

for 5minutes. The ethanol was decanted.

2.5% sodium hypochloride solution and few drops of TWEEN 20 was

added to the explants in a culture vessel and covered; it was then

placed on electric shaker to shake vigorously for 20minutes. This

enables the penetration of the sterilants.

The bleach was decanted and the explants were washed severally

with sterile distilled water.

Swab the surface of the lamina air flow with cotton wool soaked in

70% ethanol and also swab your hands with the 70% ethanol.

The explants were put into 70% ethanol in a culture vessel and

covered; it was then placed on electronic shaker to shake vigorously

for 5minutes. The ethanol was decanted.

2.5% sodium hypochloride solution and few drops of TWEEN 20 was

added to the explants in a culture vessel and covered; it was then

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placed on electric shaker to shake vigorously for 20minutes. This

enables the penetration of the sterilants.

The bleach was decanted and the explants were washed severally

with sterile distilled water.

Swab the surface of the lamina air flow with cotton wool soaked in

70% ethanol and also swab your hands with the 70% ethanol.

Using flamed forcep collect the explants from the culture vessel and

place in sterilized Petri dish, cut into nodal segments (remove the

outer edges that was assumed to have been damaged by the

sterilants).

Then the explants were inoculated into the artificial medium and

sealed with paraffin to avoid contamination by pathogens.

These inoculated explants were taken to the culture room and was

allowed to stay for 16hrs photoperiod and 8hrs dark period of

photosynthesis at a temperature of 280c +or-2, 50-60% relative

humidity.

After defined period (6weeks) shoots/roots will develop from the

explants.

STAGE III: Multiplication of the Shoots by Series of Sub-culturing

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At this stage the in vitro plantlet is re-divided and placed in a fresh

medium so as to produce more plantlet. This process is repeated many

times until the number of plants desired is reached.

Aim: The essence of sub-culturing is for the multiplication of the plantlets,

and due to the exhaustion of the nutrient and dehydration of water by

introducing them in a freshly prepared nutrient medium.

Materials/Equipments/Apparatus: Petri dishes, surgical blade, scalpel

and its handle, lamina air flow, paraffin, ethanol etc

Procedure:

Swab the surface of the lamina air flow with cotton wool soaked in

70% ethanol and also swab your hands with the 70% ethanol.

The scalpel, forcep were flame sterilized in a glass bid sterilizer i.e.

dipping them in 70% ethanol, followed by flaming in the sterilizer

and cooling on the lamina air flow/hood before use. This is called

incineration.

The young explants were cut into subcultures and inoculated into

freshly prepared medium so as to produce more plantlets.

Always note the direction of the node, so that it is not turn upside

down when inoculating.

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The inoculated subcultures were taken to the culture room where

they were kept until the time of weaning.

STAGE IV: Weaning/Hardening

Establishment of plantlets ex vitro; it is a gradual exposure of

plantlets for acclimatization to environmental conditions. This stage is

characterized by preparation of propagules for successful transfer to soil.

Immediately the required numbers of plantlets are generated and the

plantlets have developed a root system through regeneration, the plantlets

were removed from an artificial controlled environment where they have

been undergoing heterotrophism and exposed them to outdoor

environmental conditions (autotrophism). This is to facilitate the

acclimatization of the plantlet to field condition.

Aim: To prepare plantlet to be able to adapt to natural environment by

moving them from heterotrophic to autotrophic mode of life.

Materials/Equipments/Apparatus: peat pellets, vermiculite or crushed

coconut fiber, bowl, water, perforated white small polyethylene bag

(substrate bag), humidity chamber, rope, masking tape etc

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Procedure:

Humidity chamber was prepared and kept behind. The peat pellet

and vermiculite were soaked and mixed together in a big bowl and

then half-fill the substrate bag with it.

The plantlets were brought out from the culture room and were

labeled. The plantlets in culture vessels were gently removed and

poured into a basin of water to wash off the medium and then

transferred to substrate bag properly.

After transplanting the plantlets into the substrate bag, the bag was

then sent to the humidity chamber immediately and sprayed water

mixed with fungicides. The humidity chamber was closed and hung in

the screen house.

After 3 days, the humidity chamber was punctured and watering of

plantlets at least twice a day started on the fourth day.

These plantlets were allowed to stay in the humidity chamber with

constant watering for 4-6weeks before they were transferred to

polyethylene bags of sterilized top soil (top soil bag).

The plantlets stayed for 6weeks in the screen house before they

were transferred to the field.

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STAGE V: Planting Stage

This is a stage where the plantlets were transferred from the screen

house to the field to be planted directly to the soil i.e. finally exposed to the

natural environment.

There are usually two types of transplanting methods used:

Ball earth transplanting: This is planting the plantlets with the soil

below it.

Naked root transplanting: This is planting the plantlets without the

soil below it e.g. the roots are planted nakedly into the ground.

2.4 MEASURES TAKEN TO MAINTAIN ASEPTIC CONDITION IN THE

TISSUE CULTURE LABORATORY

During in vitro cultures, maintenance of aseptic environment is the

most difficult task, because the cultures can easily be contaminated by

fungi and bacteria present in the air, hence sterile conditions are required

for this process.

Some measures taken to maintain aseptic condition in the laboratory are:

Provision of an Anteroom: this is a small room where staffs

changes their foot wears before entering the laboratory and also to

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prevent the inflow of air from natural environment so to reduce the

introduction of pathogen into the laboratory.

Use of chemical sterilants e.g. 70% ethanol, 2.5% sodium

hypochlorite.

Autoclaving: The medium is sterilized after preparation in an

autoclave at the temperature of 1210c, 1.05kg/cm3, for 15minutes

while sterilization of glass wares is for 40minutes.

Sterilization of water used in the laboratory.

Laboratory coats are worn always, once inside the laboratory.

Any person suffering from cough and catarrh are not allowed inside

the laboratory to avoid cross contamination.

The culture room is strictly out of bound to unauthorized person(s).

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CHAPTER THREE

3.1 MOLECULAR BIOLOGY LABORATORY

Molecular biology is the branch of biology that deals with the

structures and functions of macromolecules (e.g. protein and nucleic) that

are essential to life. The sole aim of this laboratory in NRCRI is the genetic

improvement of cassava cultivars’ and other cultivars grown in NRCRI

through molecular breeding. We base on the following techniques: DNA

extraction from plant, running of Agarose Gel Electrophoresis and PCR.

3.2 BASIC EQUIPMENTS IN MOLECULAR BIOLOGY LABORATORY

Centrifuge: This is a machine that is used in the laboratory to spin a

sample at a high speed. The centrifugal force pulls the plant leaves to

the bottom of the dilution tubes, separating the liquid by sending it to

the top.

Lyophilizer: This can also be called freeze-dryer. It is a dehydration

technique, which enables liquid or slurry products, which have

previously been frozen to be dried under a vacuum. Works at -400c.

Tissue lyser: This enables you to homogenize, lyse up to 24 samples

at a time. Simply place your sample, tungsten carbide beads, and

buffer in a collection tube, and load the tubes into the bullet blender

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homogenizer. Adjust the run time and speed. Afterwards, place the

sample tubes in a centrifuge to separate the lysate.

Refrigerator (-800c): this equipment is used to store samples to

avoid drying up before taking them to freeze-dryer.

3.3 ACTIVITIES IN MOLECULAR BIOLOGY LABORATORY

I. DNA extraction

DNA extraction is the removal of deoxyribonucleic acid from the cells

or viruses in which it normally resides. DNA is a negatively charged

molecule that encodes the genetic information in the cell and is capable of

self-replicating and synthesis of RNA.

Aim: Extraction of DNA from plants for research purposes; to determine

the gene that is responsible for a particular trait.

Materials/Equipments/Apparatus: Tissue lyser, freeze dryer, centrifuge,

elution tube, dilution tube, S-block, Dneasy 96 plate, pipette, tungsten

carbide beads etc.

Notes before starting:

This protocol is for purifying DNA from 2x 96 samples of fresh plant

tissue.

Add ethanol to Buffer AW1 and Buffer AW2 concentrates.

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Preheat Buffer AP1 to 650c.

Combine 90ml Buffer AP1, 225microliter RNase A and 225microliter

Reagent DX.

Procedure:

The samples were collected from the field (apex of a plant) and were

stored in the freezer at -800c.

When brought from the freezer, it was transferred to freeze dryer to

dry up the samples (it works at -400c).

Place up to 50mg leaves into each dilution tube in 2 collection

microtube racks.

Add 2 tungsten carbide beads to each collection microtube and

pipette 400µl of buffer AP1 (lysis buffer) transfer it into each

collection microtube. Tightly seal the microtubes using strip caps.

Assemble each rack of collection microtubes into the tissue lyser.

Grind the sample for 1.5minutes at 30Hz.

Centrifuge to collect any solution from the caps.

Add 130µl buffer P3 (neutralization buffer) to each collection

microtube and reseal using new caps.

Place a clear cover over each rack and shake vigorously up and down

for 15s. Centrifuge to collect any solution from the caps.

Incubate the collection microtube racks for 10minutes at -200c.

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Centrifuge the collection microtube racks for 5minutes at 3800 x g

(6000rpm).

Transfer 400µl of each supernatant to a new collection microtube.

Add 600µl of buffer AW1 (washing buffer) to each sample. Close

microtubes with new caps.

Place a clear cover over each rack and shake vigorously up and down

for 15s. Centrifuge to collect any solution from the caps.

Place 2 DNeasy 96 plates on top of S-Blocks. Mark the DNeasy 96

plates for later sample identification.

Transfer 1ml of each sample to each well of the DNeasy 96 plates.

Seal each DNeasy 96 plate with an AirPore Tape Sheet. Centrifuge for

4minutes at 3800 x g.

Remove the tape. Add 800µl of buffer AW2 (washing buffer) to each

sample. Centrifuge for 5minutes at 3800 x g without tape to dry the

membranes.

Place each DNeasy 96 plate on a new Elution Microtubes RS rack.

Add 100µl Buffer AE (washing buffer) and seal with new AirPore

Tape Sheets. Incubate for 1minute at room temperature (200c).

Centrifuge for 2minutes at 3800 x g.

Repeat the last step. Seal the Elution Microtubes RS with new caps to

store DNA.

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Precautions:

When transferring the samples into dilution tubes, it was ensured to

swab the shopping bond and the handle with distilled water and

ethanol, before using it for other specie to avoid mix up of samples.

After addition of each buffer and after using each strip cap once, it

was ensured to cover with new strip caps to avoid mix up of samples.

I ensured I was putting on lab coat and hand glove.

II. Polymerase Chain Reaction

Polymerase Chain Reaction is a revolutionary method developed by

Kary Mullis in the 1980s. PCR is a laboratory technique used to make

multiple copies of a segment of DNA. With this technique, a target sequence

of DNA can be amplified a billion fold in several hours.

Basic Principles of the PCR

The cycling reactions: there are 3 major steps in a PCR, which are

repeated for 30 or 40 cycles. This is done on an automated cycler, which

can heat and cool the tubes with the reaction mixture in a very short time.

Denaturation at 940c: During the denaturation, the double strand

melts open to single stranded DNA, all enzymatic reactions stop; e.g.

the extension from a previous cycle.

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Annealing at 540c: The primers are jiggling around, caused by the

Brownian motion. Ionic bonds are constantly formed and broken

between the single stranded primer and the single stranded

template. The more stable bonds last a little bit longer (primers that

fit exactly) and on that little piece of double stranded DNA (template

and primer), the polymerase can attach and starts copying the

template. Once there are a few bases built in, the ionic bond is so

strong between the template and the primer, that it does not break

anymore.

Extension at 720c: This is ideal temperature for the polymerase. The

primers, where there are a few bases built in, already have a stronger

ionic attraction to the template than the forces breaking these

attractions. Primers that are on positions with no exact match get

loose again (because of the higher temperature) and don’t give an

extension of the fragment.

The bases (complementary to the template) are coupled to the

primer on the 3I side (the polymerase adds dNTPs from 5I to 3I,

reading the template from 3I to 5I side, bases are added

complementary to the template). Because both strands are copied

during PCR, there is an exponential increase of the number of copies

of the gene. Suppose there is only one copy of the wanted gene before

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the cycling starts, after one cycle, there will be 2 copies, after two

cycles, there will be 4 copies, and three cycles will result in 8 copies

and so on.

III. Running of Agarose Gel Electrophoresis

Agarose gel electrophoresis is a method used in molecular biology to

separate a DNA according to their molecular sizes in a matrix of agarose,

for visualization and purification.

In gel electrophoresis, the molecules to be separated are pushed by

an electrical field through a gel that contains small pores. The molecules

travel through the pores in the gel at a speed that is inversely related to

their lengths. This means that a small DNA molecule will travel a greater

distance through the gel than will a larger DNA molecule.

Gel electrophoresis involves an electric field; in particular, this field is

applied such that one end of the gel has a positive charge and the other end

has a negative charge. Because DNA is a negatively charged molecule, it will

be pulled towards the positively charged end of the gel.

Aim: To separate and analyze DNA. The purpose of the gel might be to look

at the DNA, to quantify it or to isolate a particular band.

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Materials/Equipments/Apparatus: gel tray, gel tank, gel comb, UV light

machine, electrophoresis power supply, PCR plate, conical flask etc.

Procedure:

Measure out 1g of agarose gel sample and mix it with appropriate

buffer (50ml of 0.5x TAE/TBE) in conical flask.

Heat (microwave) for 2minutes until the agarose is completely

dissolved, then leave it to cool on the bench for 5minutes down to

about 600c.

Add 2µl of ethidium bromide and swirl to mix.

Note: The reason for allowing the agarose to cool a little before this step is

to minimize production of ethidium bromide vapour. Ethidium bromide is

mutagenic and should be handled with extreme caution. Ethidium bromide

binds with DNA and allows you to visualize the DNA under ultraviolet (UV)

light.

Pour the gel slowly into the gel tray with the gel comb in place

(benefit of pouring slowly is that most bubbles stay up in the flask.

Rinse out the flask immediately).

Leave to set for at least 30minutes, preferably 1hour, until it has

completely solidified.

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Once solidified, remove the gel comb and place the gel tray in the gel

tank, then pour 0.5x TAE/TBE into the gel tank to submerge the gel

to 2-5mm depth.

Load your samples to PCR plate, there mix it with loading dye.

Transfer them to the gel tray.

Note: Loading dye provides a visible dye that helps with gel loading and

will also allow you to gauge how far the gel has run while you are running

your gel. It also contains a high %glycerol, so after adding it, your sample

will be heavier than water and will settle to the bottom of the gel well,

instead of diffusing in the buffer.

Cover the gel tank and switch on the power source. Run the gel at

70V until the dye line is approximately 75-80% of the way down the

gel.

Turn off the power and disconnect the electrodes from the power

source.

Carefully remove the gel tray from the gel tank. Transfer to UV light

and visualize your DNA fragments.

Precautions:

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When working with ethidium bromide which is a known mutagen

and also UV light, I made sure I was putting on safety goggles or face

mask, hand gloves and lab coat.

CHAPTER FOUR

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4.1 TRAIT PROFILE LABORATORY

This laboratory in NRCRI deals with the extraction of starch from

different species of cassava so to determine the quantity of starch extracted

from each species.

Starch is a polysaccharide consisting of a large number of glucose

units joined by glycosidic bonds; it is found especially in seeds, tubers,

roots, stems etc. These polysaccharides are produced by most green plants

as an energy store. It is an important constituent of human diet and is

contained in large amounts in some foods like cassava, potatoes, maize

(corn), rice, wheat etc.

Pure starch is a white, tasteless and odorless powder that is insoluble

in cold water or alcohol but soluble in hot water. Dissolving starch in warm

water gives wheat paste, which can be used as a thickening or stiffening

agent. It can be applied to parts of some garments before ironing to stiffen

them. It is used in food thickener and it is also used as a substitute for some

beauty and health products like powder etc.

For cassava, the process of starch extraction is relatively simple as

there are only small amounts of secondary substances, such as protein, in

the roots. When cassava roots are harvested or selected for starch

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extraction, it needs to be processed almost immediately after the harvest,

as the roots are highly perishable.

Aim: To determine the quantity of starch in different species of cassava.

Materials/Equipments/Apparatus: blender, 150 micron sieve, knife,

chopping board, different cassava genotypes, refrigerator, water, bowl,

beaker, oven, weighing balance, etc.

Procedure:

Simple process for cassava starch production

Cassava roots

Peeling

Grinding

Settling/decantation

Cassava starch

Sieving

Washing

Chopping

Drying

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The five main stages of extraction of starch from fresh cassava roots:

Preparatory stage: here, the samples were harvested from the field,

and was taken to the laboratory where it was peeled and washed.

Chopping: The cassavas were cut into small pieces with repeated

sharp blows using knife for easy grinding.

Weighing of samples: here, the weight of the empty beaker was

taken using analytical weighing balance, after which the weighing

balance was adjusted to zero to avoid errors, and the samples were

added into the beaker and 100g was weighed for each cassava

genotype.

Blending: the cassava samples were made into solution by blending

using appropriate quantity of water for easy grinding.

Sieving: the cassava solutions are sieved using an appropriate

quantity of water, to extract the starch and remove the fiber. After

which the cassava solutions was allowed to settle and the

supernatant liquid was carefully decanted living the solid sediment

(starch) in the beaker.

Drying: the starch was dried in the oven for 24-48hrs at low

temperature of about 500c. Then the weight of starch after drying

was taken by weighing using an analytical weighing balance.

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After the extraction of starch, the quantity of starch in different

species of cassava was determined by; the weight after drying minus

the weight before drying divided by the weight of sample multiply by

100%. Mathematically:

Precaution:

Being a quantitative analysis, it was ensured that the quantity of

starch in each cassava genotype was accurate.

It was ensured that each step of the starch extraction process was

carefully carried out e.g. it was ensured to wash off the sieve, bowl

and also rinse my hands after sieving each of the sample to avoid mix

up of the samples.

weight after drying – weight before drying

weight of sample

100%

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CHAPTER FIVE

5.1 RELEVANCE OF THE SIWES PROGRAMME

The Student Industrial Work Experience Scheme (SIWES) programme

made me to understand more and recall what I had been taught. It was a

nice and interesting experience which has really exposed me to what I will

face even after my graduation and when I join the labour market.

It has brought focus to the student on the area of their specialization and

the capacity to handle the work.

5.2 PROBLEMS ENCOUNTERED

I encountered some problems during the SIWES programme though; I did

not allow them to affect me;

-The distance from my working place is far from my residence. Most times,

I starve to save money that will take me back home.

The idea of not paying IT student at least their transportation fare is not

good and encouraging.

-The problem of lack of cooperation by some of the staff to students.

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5.3 RECOMMENDATION

-Since SIWES has come to stay, Government and the board (ITF) in charge

of SIWES should assist the student by writing to various companies and

established to be accepting student for SIWES programme and improve in

the stipend aspects of it because most student stay away from their home

and school thereby spending much of their time and money in the course of

attending the programme. So stipend will even hasten up their moral to

work.

-Schools should make sure that student apply to industries that are related

to their course.

5.4 CONCLUSION

Students Industrial Work Experience Scheme (SIWES) gave me the

opportunity to practicalize the theoretical knowledge which I acquired in

the university. It really exposed me to work situation and rules and

regulations which abide a working environment.

This training has exposed me to professional ways of working, work safety

in industrial environment, and orderliness etc. through the experience, I

have acquired skills relevant to my course of study.

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Finally, SIWES programme has come to stay and is there for good.

Therefore, the future participants should try their best in utilizing and

achieving the optimum aim of this programme.