Institute for developing science and health initiatives.

1. IN-PLANT TRAINING
ON
DEPARTMENT OF BIOCHEMISTRY & MOLECULAR BIOLOGY
UNIVERSITY OF RAJSHAHI
Examination Roll: 1511125162
Registration No: 1511125162
Session: 2014-15

2. INSTITUTE FOR DEVELOPING SCIENCE AND HEALTH INITIATIVES
The initiative for establishing “Institute for Developing Science and Health Initiatives (ideSHi)” was possible with
the Christophe Rodolfe Grand Prize awarded to Dr. Firdausi Qadri in June 2012.
The institute is now located in the premises of the Centre for Medical Biotechnology of the Directorate General of
Health Services, Institute of Public Health in Dhaka. It is furnished with BSL-2 facilities to carry out molecular,
immunological, genetic and metabolic research.
Collaborating scientists from USA, Canada and France are working with ideSHi. The institute is also running training
courses for medical personnel, clinicians and biomedical researchers by focus on areas of biomedical sciences and
biotechnology where a crucial need and a major gap exists in Bangladesh and in the developing world.
Although ideSHi principally aims at introducing studies on inborn errors of metabolism (IEM) including diagnosis of
IEM, focus is also given on other genetic disorders including Down Syndrome, hereditary neuro-degenerative diseases
like Huntington’s disease, congenital hypothyroidism, thalassemia etc. as well as on infectious diseases including
respiratory infections, Hepatitis E virus infections, rapid diagnosis of typhoid fever, molecular diagnosis of ETEC
diarrhea and diarrheal diseases, as well as molecular mechanisms of antimicrobial resistance in acute respiratory and
enteric infections.

3. Mission
Of
ideSHi
.
Their mission includes:
• Creating awareness
• Providing solutions to public health problems
through translation of innovative ideas into
technologies
• Accelerating studies
• Serving as a hub for enabling research and
training activities in Bangladesh
• Provide a base for collaboration with international
research institutions to carry out studies in
Bangladesh
• Building up local capacity in the fields of
biomedical sciences and biotechnology
• Providing opportunities
• Providing financial support for students and
researchers in the form of fellowships,
scholarships and stipends.

4. Objectives
of
In-Plant Training
Program
Our perspective of this In-
plant training by the
followings:
• To learn how a Research Institute runs
• To learn how a laboratory can maintain BSL-2 &
BSL-3 facilities
• To learn how Molecular & Genetically basis
research and development performed
• To learn how different specific instruments can
use in different laboratory purpose
• To learn how everything works in an Advanced
Research Institute
In short, to prepare ourselves to perform different
task in a Research Institute.

5. Introduction to
Advanced Training
on
Molecular
Techniques
During this training program we
have observed various
techniques of Advanced
Molecular Biology such as:
• DNA extraction from blood by FlexiGene kits
• DNA and RNA quantification by Qubit-4
• RNA isolation by TRIAzol reagent
• Polymerase Chain Reaction (PCR)
• Gradient PCR by BioRad Thermal Cycler
• Real time PCR followed by HRM
• Agarose Gel electrophoresis (1.5% gel)
• PCR Product Purification by QIAquick PCR
product purification kit

6. DNA Extraction
from
Human Blood
(FlexiGene DNA extraction Kit)
FlexiGene kits:
• FlexiGene DNA Kits provide a rapid and simple
method for purification of DNA from human
whole blood, buffy coat, and cultured cells.
• FlexiGene DNA Kits provide good yields of
high-purity DNA that is free from contaminants
or inhibitors.
• The procedure can be scaled up or down,
allowing purification from variable amounts of
starting material. Purification is performed in a
single tube, which reduces waste and minimizes
the risk of sample mix-up.
• The purified DNA performs well in a range of
downstream applications including PCR-based
techniques, restriction digestion, blotting, and
sequencing, or can be safely stored at 2–8°C or
–30 to –15°C.

7. Protocol :
1. Add 500µl FG1 Buffer +200µl of whole blood.
2. Mix by inverting the tube 5 times.
3. Centrifuge 20 seconds at 10000x g.
4. Discard the supernatant.
5. Invert the tube on a clean sheet for 2 minutes.
6. Add 200µl FG2 buffer/protease mixture (FG2: Protease = 100:1)
7. Immediately vortex until the pellet is completely homogenized.
8. Centrifuge the tube briefly (3-5 sec).
9. Place it in water bath at 65°C for 5 minutes (red>green).
10. Add 100µl of isopropanol (100%).
11. Mix by inversion until DNA precipitate becomes visible as threads or a clump.
12. Centrifuge for 3 minutes at 10000xg.
13. Discard the supernatant.
14. Invert the tube onto a clean tissue.
15. Add 100µl 70% ethanol.
16. Vortex for 5 seconds.
17. Centrifuge for 3 minutes at 10000xg
18. Discard the supernatant.
19. Invert the tube on a paper for 5 minutes.
20. Air-dry the DNA pellet for 5 minutes.
21. Add 200µl Nuclease free water.
22. Store the sample at -20°C.
FlexiGene DNA
Extraction
Procedure

8. DNA and RNA Quantification by Qubit-4
• This user-friendly fluorometer delivers results
using quick, simple assay procedures. The large,
color touch screen allows you to easily navigate
through the various options.
• The Qubit-4 Fluorometer will save the data for
up to 1,000 samples, and you can transfer data
using Wi-Fi, a USB drive, or direct connection
with a USB cable. In addition, new Qubit-4
includes a built-in reagent calculator to easily
determine the amount of dye and buffer
required to prepare your samples and standards.
Figure : Qubit-4 fluorometer
Figure : Qubit-4 Fluorometer workflow

9. RNA isolation
using
TRIzol reagent
• The isolation of RNA with high quality is a crucial step
required to perform various molecular biology experiment.
• TRIzol Reagent is a ready-to-use reagent used for RNA
isolation from cells and tissues. TRIzol works by maintaining
RNA integrity during tissue homogenization, while at the
same time disrupting and breaking down cells and cell
components.
• Addition of chloroform, after the centrifugation, separates
the solution into aqueous and organic phases. RNA remains
only in the aqueous phase.
• After transferring the aqueous phase, RNA can be recovered
by precipitation with isopropyl alcohol.
• Precipitation with ethanol requires DNA from the
interphase, and an additional precipitation with isopropyl
alcohol requires proteins from the organic phase.
• Total RNA extracted by TRIzol Reagent is free from the
contamination of protein and DNA.
• This RNA can be used in Northern blot analysis, in vitro
translation, poly (A) selection, RNase protection assay, and
molecular cloning.
RNA isolation using TRIzol reagent
:

10. PROTOCOL
1. Add 500µl of Isopropanol to the acquous phage per 750 µl of TRIzol is
Reagent used for analysis.
2. Incubate for 10 minutes.
3. Centrifuge for 10 minutes at 12000 xg at 4ºC.
4. Discard the supernatant.
5. Resuspend the pellet in 1ml of 75% ethanol per 750 µl of TRIzol LS
Reagent.
6. Vortex the sample briefly, then centrifuge for 5 min at 7500xg at 4ºC
7. Discard the supernatant.
8. Vacum or air dry the RNA pellet in 20-50 µl of RNase free water ,0.1
mM EDTA or 0.5% SDS solution by pipetting up and down.
9. Incubate in a water bath or Heat block set at 55-60ºC for 10-15
minutes.
** Store the RNA at -70ºC
STEP-2: Isolate the RNA
STEP-1: Lyse samples and separate phase
1. Add 750µl of TRIzol LS reagent per 250µl of sample volume.
2. Homogenize the ample by pipetting up and down several tim.
3. Centrifuge the lysate for 5 minute at 12000 xg at 4-10ºC, ransfer the clear
supernatant to a new tube
4. Incubate 5 minutes.
5. Add 200µl of chloroform per 750µl of TRIzol LS used for lysis.
6. Incubate 2-3 minute.
7. Centrifuge the sample 15 minute 12000 xg at 4ºC
8. Transfer the colourless upper aquous phase containing the RNA to a new
tube.
9. Transfer the aquous phase containing the RNA to nee tube by Angling the at
45º nad pipetting the solution out.

11. Acknowledgements
Dr. MD. Khaled Hossain
Professor
M.Sc. (RU), PhD (Japan)
Research Interest:
Environmental Health Science, Effects
of Arsenic and Other Heavy Metal on
Human Health, Immunology
Dr. Imtiaj Hasan
Associate Professor
M.Sc. (RU), MPhil (RU), PhD ( YCU, Japan)
Research Interest:
Glycobiology, Protein, Biochemistry,
Microbiology, Anticancer Drug
Development, Multidrug Resistance
Dr. Jahan Ara Khanam
Professor
M.Sc. (RU), PhD (India)
Research Interest:
Oncomedicine and Anticancer Drug
Design

12. THANK YOU