Upload
henrietta-ezeagu
View
307
Download
5
Tags:
Embed Size (px)
Citation preview
1
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
2
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.
3
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).
4
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
5
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
6
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.
7
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
8
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.
9
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
10
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.
11
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
12
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
13
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.
14
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.
15
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.
16
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
17
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
18
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
19
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:
20
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).
21
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
22
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
23
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.
24
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
25
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.
26
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
27
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).
28
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
29
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.
30
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.
31
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.
32
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.
33
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
34
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.
35
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.
36
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:
37
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
38
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
39
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
40
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.
41
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%
42
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.
43
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.
44
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.