This document is a presentation by Onyiba Cosmos Ifeanyi on their Students’ Industrial Work Experience Scheme (SIWES) done at the International Institute of Tropical Agriculture (IITA) in Ibadan, Nigeria. It summarizes the trainings received, including tissue culture, cassava seed production via pollination, and nucleic acid extraction. Methods for cassava pollination and total nucleic acid extraction using the CTAB method are described. The presentation also lists the instruments used such as a vortex, shaker, centrifuge, thermocycler and gel doc.

1. A
Presentation on
Students’ Industrial Work Experience Scheme (SIWES)
done at Harvestplus c/o International Institute of Tropical
Agriculture (IITA), PMB 5320, Oyo Road, Idi-Oshe, Ibadan, Nigeria
Presented
By
ONYIBA, Cosmos Ifeanyi
MOUAU/10/15593
Department of Biochemistry
College of Natural and Applied Sciences
Micheal Okpara University of Agriculture, Umudike
March, 2014
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Onyiba Cosmos I.
cosmos_onyiba@yahoo.com
+2348062633518

2. Trainings Undergone
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Tissue culture
Cassava seed production via pollination
High quality vitamin A cassava processing
Cassava pests and disease control
Extraction of Total Nucleic acid (DNA and RNA) from cassava
Enzyme-linked Immunosorbent Assay (ELISA)
Mechanical sap inoculation of plant viruses
Polymerase Chain Reaction (PCR)
Gel Electrophoresis
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org

3. Cassava seed production
pollination
via
Steps involved
The male and female flowers of
interest are first identified. The
female flowers are bagged
The male flowers are collected in
clean sterile bags fully labelled and
kept in cold environment
The female flowers are unbagged at
mid-day. Pollen is dusted into the
stigma. The crosses made is labelled in
a tag.
The pollinated flowers are bagged
and unbagged after 72 hours and
allowed to grow into fruits.
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org

4. Pollination continued…
 Fruit develop after 45-72 days.
mature fruit turn from dark green to
pale.
 Mature fruits are collected Into
bags, sun dried and allowed to shatter
 The Dried Fruits Are Shelled To
Obtain Seeds.
 Seeds obtained are stored in
labelled envelopes and kept in cold
environment
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org

5. TOTAL NUCLEIC ACID EXTRACTION AND PURIFICATION
CTAB METHOD
CTAB buffer compositionKEY COMPONENTS OF EXTRACTION
20mM Ethylenediaminetetra Acetic
Acid.
100mM Trizma Base.
1.4M Sodium Chloride.
0.2% 2- Mercaptolethanol.
Sampling: Samples mainly collected are cassava leaves.
Lysis: The leaves are grinded in CTAB buffer
Removal of Protein and contaminants : Phenol-
Chloroform and Isoamyl- alcohol are combined to get
rid of proteins and other contaminants. The isoamyl-
alcohol prevents foaming effect when phenol denatures
protein.
Precipitation: Isopropanol stored in -200C is used to aid
pellet (DNA/RNA) formation and remove salts from
pellet while ethanol is used to further purify the pellet.
Recovery: The pellet is then resuspended in sterile water
and stored in -200 or used for further analysis
e.g PCR, Gel Electrophoresis.
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org

6. TOTAL NUCLEIC ACID EXTRACTION
CTAB METHOD
Lysis of cell wall and membrane
(Aqueous phase)
supernatant containing
nucleic acid
Centrifugation
(Organic phase) containing
proteins and other
contaminants
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Removalofproteinand
contaminants
Chloroform,phenoland
isoamylalcohol

7. TOTAL NUCLEIC ACID EXTRACTION AND PURIFICATION
CTAB METHOD
DNA Precipitation and
Recovery
Nucleic acid+ Isopropyl
Alcohol
Centrifugation
Washing with ethanol Refrigeration at -800 C for 15
minutes
The pellet is suspended in
sterile water
DNA/RNA pellet
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org

8. INSTRUMENTATION
The VortexThe shakerThe centrifuge
The ELISA Reader and Printer The Gel electrophoresis machine
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org

9. INSTRUMENTATION
The Thermocycler The pH meter
The Nanodrop SpectrophotometerThe Gel doc (Biorad)
International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org

10. International Institute of Tropical Agriculture – Institut international d’agriculture tropicale – www.iita.org
Onyiba Cosmos I.
cosmos_onyiba@yahoo.com
+2348062633518