INTERNSHIP REPORT

Internship at National Institute of Biotechnology & Genetic Engineering (NIBGE) Faisalabad

From 17th July to 17th August, 2023

Internship work conducted under supervision of
DR. Naveed Altaf Malik
(Principal Scientist)
Submitted to:
DR. Naveed Altaf Malik (P.S)
Submitted By:
Sumera Shaikh D/O Umer-din
Registration No: 2k20-Gen-56
Roll No: 2k20-Gen-56
Session: 2020-2023
Semester: 8th (Final Semester)
 CERTIFICATION
It is certified that Sumera Shaikh D/O Umer-din Reg. No: 2k20-Gen-56 is
a student of Genetic Engineering at Institute of Biotechnology & Genetic
Engineering (IBGE), University of Sindh, Jamshoro. This report is submitted by her
satisfactory to fulfill the requirements of degree.

Supervisor:
Dr. Naveed Altaf Malik
Principal Scientist, Health
Biotechnology Division,
NIBGE, Faisalabad
 ACKNOWLEDGEMENTS

I am immensely grateful for the invaluable opportunity to complete a transformative one-week internship
at the human Molecular genetics lab HMG, Cytogenetic/karyotyping Lab in the health biotechnology
division; also exert at nanobiotechnology lab (synthesis of nanoparticle), Biotechnology of Prebiotic and
antimicrobial Lab in the division of Industrial biotechnology, located within the esteemed National institute
of biotechnology and genetic engineering in Faisalabad.
I extend my sincere thanks and praise to the Almighty for granting me the knowledge, wisdom, and
perseverance that allowed me to make the most of this experience.
First and foremost, I would like to express my heartfelt appreciation to Director Dr. Kulsoom Akhter of
NIBGE, also thanks Dr. Muhammad Rafique director of institute of Biotechnology and Genetic
Engineering University of Sindh, Jamshro and Supervisor Dr. Naveed Altaf Malik (PS) for providing me
with this chance to learn and grow in such a prestigious Research Center. Their belief in my potential and
unwavering support throughout the internship were instrumental in making this journey so rewarding.
I am indebted to MR. Naeem and Dr. Abdul Ghafar for their exceptional guidance, support, and mentorship
during my internship in health division and also thanks to Dr. Iqra Jawad, Mr. Abdul Mateen for their
expertise and encouragement played a pivotal role in shaping my professional growth and learning journey
in Biotechnology of prebiotic and antimicrobial lab at Industrial biotechnology Division.
My gratitude also extends to Dr.Sadia Zafar bajwa, Mr. Daim for their kind and support throughout this
period in nanobiotechnology Lab at industrial biotechnology division. Their encouragement and assistance
enabled me to grasp techniques swiftly and make the most of the learning opportunities. I would like to
acknowledge and express my sincere thanks to Dr. Abdul Malik and Dr. Nasir Ahmad for providing me
with this invaluable opportunity to intern at NIBGE. Their belief in my potential opened doors to a world
of knowledge and practical experiences that will undoubtedly serve as a strong foundation for my future
career endeavors.
The internship experience at NIBGE has been truly transformative, leaving me with a sense of confidence
and enthusiasm for my chosen field. The skills and knowledge I have acquired during this period will
undoubtedly play a pivotal role in shaping my future endeavors.
In conclusion, I express my heartfelt appreciation to the entire team at NIBGE for the support, guidance,
and knowledge shared during my internship. I am eager to stay connected and contribute positively to the
Research Center whenever possible, continuing our shared pursuit of scientific excellence and
advancement.
Sumera Shaikh
 ABSTRACT
This report provides a comprehensive summary of the skills I gained during my one week internship at
NIBGE, Faisalabad. I utilized my skills in many different labs where I learnt investigates the intricate links
between genetic variations and human health. Utilizing advanced techniques like PCR and gene expression
analysis, the lab identifies genetic mutations responsible for inherited diseases. By unraveling molecular
pathways, this research contributes to understanding disease susceptibility and informs personalized
medical interventions in Human Molecular Genetics Lab. Also analysis the detection of chromosomal
aberration, genetically linked disease by the help of karyotyping. Nanobiotechnology is gaining tremendous
impetus in this era owing to its ability to modulate metals into their nanosize, which efficiently changes
their chemical, physical, and optical properties. I also learnt about new field of prebiotics, Prebiotics are a
group of nutrients that are degraded by gut microbiota. Their relationship with human overall health has
been an area of increasing interest in recent years. They can feed the intestinal microbiota, and their
degradation products are short-chain fatty acids that are released into blood circulation, consequently,
affecting not only the gastrointestinal tracts but also other distant organs. Fructo-oligosaccharides and
galacto-oligosaccharides are the two important groups of prebiotics with beneficial effects on human health.
Consequently, my confidence in executing complex laboratory protocols has significantly improved, and I
have strengthened my ability to analyze in research and interpret experimental data. These newly acquired
techniques have become invaluable assets, preparing me for a promising future career in the field of
biological sciences.
 CONTENTS
Introduction to NIBGE
• Research Divisions
 Agriculture Biotechnology Division
 Health Biotechnology Division
 Industrial Biotechnology Division
 Soil & Environmental Biotechnology Division
Health biotechnology Division
 Human Molecular Genetics Lab
 Human Genomic DNA Extraction
 1) Phenol: Chloroform OR Organic OR chemical method
 Blood Collection Protocol
 DNA Extraction Protocol
 2) Enzymatic OR Proteinase K OR Kit Method
 Blood Collection Protocol
 DNA Extraction Protocol
 Extraction of DNA from chorionic villi (CVS)
 CVS Sample Collection Protocol
 CVS Extraction Protocol
 Gel Electrophoresis
 Agarose Gel Protocol
 Gel Electrophoresis Protocol
 Analysis
 Polymerase Chain Reaction
 Thermocycler
 Components of PCR
 PCR Mixture Preparation Protocol
 PCR Machine Protocol
 Primer
 Types of Primers
 Primer designing
 Designing PCR Primers Cautions
 Primer designing tools
 Primer designing through Ensemble
 Primer designing through Primer 3
 Primer designing through Primer Blast
 Pedigree
 Reading a Pedigree
 Primer designing
 To start reading a pedigree:
  Determine whether the trait is dominant or recessive
 Determine if the chart shows an autosomal or sex-linked (usually X-linked) trait
 Example: Autosomal Dominant Trait
 Example: X-linked recessive trait
 HaploPainter (A tool to draw pedigree)
 Purpose
 Features
 Cytogenetic/Karyotyping Lab
 Karyotyping
 Blood Collection Protocol
 Cell culture Protocol
 Harvesting Protocol
 Staining Protocol
 Screening Protocol
 DNA Extraction Protocol
Industrial biotechnology Division
 Synthesis of Nano Particles Lab-1
 Demonstration of Nano Particle Lab
 Introduction
 Definition
 Preparation of Nano Particles
 Factors Affecting the Synthesis of Nano particles
 Characterization of the synthesized Nano Particles
 Green synthesis of Nano Particles
 Method
 Tectona Grandis Seed Extract preparation
 Synthesis of Silver Nano Particles using seed extract
 Antibacterial assay
 Biotechnology of Pre-Biotic & Anti-Microbial Lab
 Introduction
 Definitions
 Criteria
 Types of Pre-Biotics
 Thin Layer Chromatography
 TLC Protocol
 Staining Protocol
 MRS Media
 MRS Media Protocol
 Petri Plate Pouring Protocol
 Inoculation Protocol
 Microscopy
 Bacterial DNA Extraction
  Bacterial DNA Extraction Protocol
Conclusion
Introduction to NIBGE
National Institute for Biotechnology and Genetic Engineering or NIBGE is one of the main biotechnology
institutes operated by Pakistan Atomic Energy Commission (PAEC). It was planned under the auspices of
PAEC in 1987 and was formally inaugurated in 1994. It is affiliated to Pakistan Institute of Engineering &
Applied Sciences (PIEAS) Islamabad, for awarding MPhil & PhD degrees. NIBGE is also the home
institution of National Biology Talent Contest. The institute is located in Faisalabad.
The institute is a focal point of modern biotechnology and provides a technology receiving unit to help the
development of country through applications of modern biotechnology and genetic engineering. The
research programs at NIBGE are mainly aimed at improving agriculture, health, environment and industry
and are supported by national and international financial grants. The institute research facilities include
state of the art equipment’s supported by technical services, IT facility and a National Library for Biological
Sciences. The institute now offers several services and marketable products. The educational programs
leading to MPhil and PhD degrees have also been incorporated in the institutes mandate for the development
of human resources in modern sciences

.
Research Divisions
There are four research divisions at NIBGE:
1. Agricultural Biotechnology Division (ABD).
2. Health Biotechnology Division (HBD).
3. Industrial Biotechnology Division (IBD).
4. Soil and Environmental Biotechnology Division (SEBD).
Agricultural Biotechnology Division
Head, Agricultural Biotechnology Division: Dr. Nasir Ahmad Saeed
Agriculture is the backbone of the economy of Pakistan. The sector employees 50% of the labour,
contributes about 22.5% to GDP and sustain major industries of the country. Pakistan has one of the fastest
population growth rates and food security is the major issue for the nation. The total population of the
country is around 220 million which is expected to rise to 250 million by 2025. The success of the Green
Revolution of the earlier decades will now have to be repeated through a “Gene Revolution”.
The Division has gathered research facilities, manpower with sufficient levels of expertise and a critical
mass essential to realize goals mentioned above. Research endeavors in Agricultural Biotechnology
Division have reached a stage that their exploitation at commercial level is becoming a reality. The program
is focused on six different crops including cotton wheat, rice, sugarcane, potato and oil-seed crops
(including soybean and canola). Several steps required to take technology to end-users have been taken that
include technology transfer to public and private entrepreneurs, patenting of technology, approval for
laboratory and field testing of genetically-modified crops by Institutional.
Biosafety Committee (IBC) and National Biosafety Committee (NBC), Ministry of Climate Change,
Government of Pakistan. A number of cotton and wheat lines have been approved by the Punjab Seed
Council while plenty of others like cotton, wheat and rice are in the pipeline. Similarly indigenously
developed insect resistant and herbicide tolerant cotton expressing two (cry1Ac, cry2Ab) and three (cry1Ac,
cry2Ab, cp4-epsps) genes have been shared with the breeders at NIAB, NIBGE and NIA. Many different
transgenic / genome edited lines having improved traits like fiber quality, biotic stress resistance (chewing
and sap-sucking insects), abiotic stress (salinity, drought, frost etc.)tolerance, nitrogen use efficiency,
disease resistance (CLCuV, late blight, bacterial blight) are in the pipeline and are at different stages of
development and testing.
Health Biotechnology Division
Head, Health Biotechnology Division: Dr. Mazhar Iqbal
Heath Biotechnology Division at NIBGE is striving for healthier Pakistan through research and
development to combat major genetic and infectious diseases. Using the modern tools of biotechnology and
genetic engineering scientists at Health Biotechnology Division are investigating the molecular basis of
infectious, genetic and metabolic disorders to establish nucleic acid based diagnostics and to design
improved cost effective therapeutic strategies for quality life. Development of modern vaccines, discovery
of drugs, structure and function studies of membrane and viral proteins are the major focus of ongoing
research projects in this division.
Tradition of consanguineous marriages and relatively high frequency of disease alleles in Pakistani
population is leading to increased incidence of genetic disorders. Disease gene mapping for various
phenotypes is in progress at this division to establish the carrier screening tests and prenatal diagnosis to
prevent the affected births. Similarly, poor hygienic conditions and limited health facilities are resulting in
high incidence of infectious diseases in this country. Projects are in progress to study the molecular basis
of drug resistance and virulence of bacterial pathogens causing major diseases such as tuberculosis and
typhoid. Molecular methods for early diagnosis and genotyping for efficient and cost effective therapy are
established by the researchers of this division to reduce the economic burden. Basic research work for the
production of conjugate vaccines for typhoid and recombinant vaccines for hepatitis B and C is underway.
Recombinant and viral vector vaccines are being developed against poultry viruses IBDV and FAdV, and
animal pathogen Pasteurella multocida. Two potential classes of organic compounds have been identified
by one of our scientist and are being pursued for inhibiting HBV and HCV.
Industrial Biotechnology Division
Head, Industrial Biotechnology Division: Dr. Kalsoom Akhter
Industrial biotechnology is a multidisciplinary technology and includes the integrated application of
disciplines such as biochemistry, microbiology, molecular genetics and process technology to develop
useful processes and products, based on microbial, animal or plant cells, their organelles or enzymes as
biocatalysts. Particularly microorganisms have received a lot of attention as a biotechnological instrument
and are used in so-called fermentation processes.
The aim of our research is to develop biological processes by using living cells (such as bacteria, yeast,
algae) or component of cells like enzymes, to create sustainable manufacturing processes and products
including enzymes, biofuels, prebiotics and antimicrobials etc. The main research activities includes,
isolation of microorganisms from nature, screening for product formation, maintenance of microbial
cultures, improvement of product yield, study of biosynthetic pathways, synthesis of nanomaterials, mass
cultures using bioreactors and recovery of products and services
These research activities are focused around four different themes.
• Bioprocessing of Ores and Fossil Fuels
• Industrial Enzymes and Biofuels
• Biotechnology of Prebiotics and Antimicrobials
• Nanobiotechnology

Soil & Environmental Biotechnology Division

Head, Soil & Environmental Biotechnology Division: Dr. Samina Iqbal
As a consequence of rapid increase in population, industrialization and urbanization, Pakistan is seriously
confronted by many complex and difficult environmental challenges related to air, water, soil pollution and
depleted natural resources. Up to 35% of the burden of disease is attributable to environment hazards and
risk factors. Mandate of Soil &Environmental Biotechnology is to carry out research and develop
technologies and production the context of environmental protection.
• Development of floating and constructed wetland technologies for treating industrial and
municipal wastewaters to protect water resources, ecosystems, and human health.
• Development of NEW integrated wastewater treatments technologies.
• Restoring sites contaminated with hazardous materials using advanced bioremediation
technologies.
• Development of environmentally benign products to be used in agriculture.
• Development of indigenously formulated bio-control agents for plant diseases e.g. bacterial
blight and brown spot of rice.
• Development of third generation bio-fertilizers by incorporating promising microbial
consortia to reduce the use of chemicals/to enhance crops production for sustainable
agriculture and environment.
• Risk assessment and toxicology, to determine potential risks caused by the introduction of
GM plants, the use of certain substances in food, feed, medicine and plant protection
products etc. and chemical contaminants present in the environment.
• Livestock genomics and diagnostics.
HEALTH BIOTECHNOLOGY DIVISION
Human Molecular Genetics Lab
Human Genomic DNA Extraction
Human genomic DNA extraction involves isolating and purifying DNA from human cells to study
its genetic information. The process typically includes cell lysis, where cells are broken open to
release their contents, followed by DNA purification to remove proteins and other contaminants.
Various methods like phenol-chloroform extraction, proteinase K or kit method techniques are
used for this purpose. Ultimately, the extracted DNA can be used for various applications such as
genetic testing, research, and forensic analysis.

1) Phenol: Chloroform OR Organic OR Chemical Method

Blood Collection Protocol:
Required Material:
 Syringe,
 tourniquet,
 blood collection tubes or vial,
 Anticoagulant,
 Alcohol or disinfectant,
 holder/Adaptor,
 Gloves,
 Gauze sponge.

Procedure:
 First explained the procedure to the patient and obtained their consent.
 Identified a suitable site for blood collection, usually the inner elbow area or the back of the hand
or peripheral vein.
 Cleaned the skin at the selected site with alcohol or a disinfectant and allowed it to dry.
 Used a needle and syringe or a vacutainer system to draw the required amount of blood from the
vein. The amount of blood collected depends on the specific DNA extraction protocol and the
intended downstream applications.
 Mixed the collected blood with the appropriate anticoagulant (e.g., EDTA) to prevent clotting and
preserved the DNA.
 Properly labeled the blood collection tube with the patient's details and any relevant information.
 Store the blood sample properly to preserve its integrity and prevent contamination.
DNA EXTRACTION PROTOCOL:

Required Material:
 Microcentrifuge tube,
 micropipette (10µl-1000µl), (20µl-200µl), (1µl-20µl),
 tissue paper,
 Vortex machine,
 Centrifuge machine,
 Thermo-mixer,
 Vacuum dryer,
 Magnetic stirrer,
 Water Bath,
 Refrigerator(4ċ).

Required Chemicals:
 Solution A,
 Solution B,
 Solution C,
 Solution D,
 10% SDS,
 Proteinase K,
 3M Sodium Acetate (pH-6),
 Isopropanol,
 70% Ethanol,
 DNA Dissolving solution Or PCR Water.

Solutions Preparation:

 Solution A Recipe:

54.72g of 0.32M Sucrose, 5ml of 10mM of Tris HCl (pH-7.5), 2.5ml of 5mM MgCl2, dissolved in 400ml
of distilled water and then adjust pH to 7.5 and raised the volume up to 495ml and then autoclaved it and
let it cooled at room temperature, then added 5ml of Triton x-100.

 Solution B Recipe:

Mixed 5ml of 10mM Tris HCl (pH-7.5), 40ml of 400mM of NaCl, 2ml of 2mm EDTA, and raised volume
up to 500ml and then autoclaved it and kept at 4ċ.

 Solution C Recipe:

Firstly melted phenol at 68ċ in water bath. Took 100ml of melted phenol in bottle and added 100mg of
0.1% Hydroxyquiniline and 100ml of 0.5M Tris HCl (pH-8), stirred for 15 minutes at magnetic stirrer.
Discarded upper phase. Repeat same step until the pH of Phenol became 7.8, then added 10ml of Tris HCl
containing 200µl of β-mercapto ethanol. Stored at 4ċ.
  Solution D Recipe:

Took 24ml of Chloroform in bottle and added 1ml of Isoamylalcohol in it and then mixed and stored
solution.

 10% SDS:

Took 10g of SDS and dissolved in 80ml of distilled water (As it do get dissolved at basic pH so while
shaking at medium hot plate added 1-2 pellets of NaOH), then adjust the pH at 7.2 by conc.HCl and then
raised the volume up to 100ml.

 3M Sodium Acetate (pH-6):

Took 8.162g of Sodium Acetate and dissolved in 10ml of distilled water with the help of magnetic stirrer,
adjust the pH at 6 and then raise the volume up to 20ml.

 70% Ethanol:

Took 70ml absolute ethanol and dissolved in 30ml of distilled water.

Procedure:
 Took 750µl of blood in 1.5ml microcentrifuge tube and 750µl of Solution A.
 Mixed it by inversion and kept the tubes at room temperature for 10 minutes.
 Centrifuge the tubes for 1 minute at 13000rpm and discarded the supernatant.
 Resuspended the pellet in 400µl of Solution A and centrifuge for 1 minute at 13000rpm.
 Discarded the supernatant and resuspended the nuclear pellet in 400µl of Solution B then added
25µl of 10% SDS and 8µl of Proteinase K.
 Left the tubes at 65ċ for 3 hours.
 After this added 0.5ml of freshly prepared equal volume of Solution C and Solution D, and mixed
them.
 Vortex for 45 to 50 seconds.
 Centrifuge for 15 minutes at 13000rpm.
 Collected the upper aqueous phase in new 1.5ml tube and added equal quality of Solution D alone.
 Centrifuge for 10 minutes at 13000rpm and again removed aqueous phase in new 1.5ml tube.
 To the collected aqueous phase, added 55µl of 3M Sodium acetate (pH 6) and equal volume of
isopropanol.
 Inverted the tubes gently to precipitated DNA.
 Centrifuge at 13000rpm for 15 minutes.
 Removed the solvent phase carefully without disturbing the DNA pellet.
 To the tube containing pallet DNA added 350µl of 70% ethanol and then centrifuge at 13000rpm
for 15 minutes.
 Removed the ethanol and dry the DNA pallet in vacuum dryer.
 Kept dried pellet at 65ċ for 30 minutes.
 Dissolved the DNA in DNA dissolving solution or PCR water.
 Stored at 4ċ for further analysis (Gel Electrophoresis or PCR).
2) Enzymatic OR Proteinase K OR Kit Method
Blood Collection Protocol:
Required Material:
 Syringe,
 tourniquet,
 blood collection tubes or vial,
 Anticoagulant,
 Alcohol or disinfectant,
 holder/Adaptor,
 Gloves,
 Gauze sponge.

Procedure:
 First explained the procedure to the patient and obtained their consent.
 Identified a suitable site for blood collection, usually the inner elbow area or the back of the hand
or peripheral vein.
 Cleaned the skin at the selected site with alcohol or a disinfectant and allowed it to dry.
 Used a needle and syringe or a vacutainer system to draw the required amount of blood from the
vein. The amount of blood collected depends on the specific DNA extraction protocol and the
intended downstream applications.
 Mixed the collected blood with the appropriate anticoagulant (e.g., EDTA) to prevent clotting and
preserved the DNA.
 Properly labeled the blood collection tube with the patient's details and any relevant information.
 Store the blood sample properly to preserve its integrity and prevent contamination.

DNA EXTRACTION PROTOCOL:

Required Material:
 Microcentrifuge tube,
 micropipette (10µl-1000µl), (20µl-200µl), (1µl-20µl),
 tissue paper,
 Vortex machine,
 Centrifuge machine,
 Thermo-mixer,
 Vacuum dryer,
 Spin Column,
 Refrigerator(4ċ).

Required Chemicals:
  Ice cold nuclease-free water,
 1X PBS,
 Proteinase K Solution,
 Lysis Solution,
 Ethanol (96-100%),
 Wash Buffer WB I,
 Wash Buffer II,
 Elution Buffer.

Procedure:
 Added 1ml of ice-cold nuclease –free water to 500µl of whole blood, then mixed thoroughly by
vortexing or pipetting
 Incubated the sample for 5-7 minutes at room temperature
 Centrifuge at 3000rpm for 5 minutes, then discard the supernatant.
 Resuspended the pellet in 200µl of 1X PBS.
 Added 20µl of Proteinase K Solution, then mixed by vortexing
 Added 400µl of Lysis Solution, then mixed thoroughly by vortexing or pipetting to obtain a uniform
suspension
 Incubated the sample at 56ċ for 45 minutes while vortexing occasionally or used a shaking water
bath, rocking platform or thermo-mixer until the cells are completely lysed
 Added 200µl of ethanol (96-100%) , then mixed by pipetting
 Transferred the prepared mixture to the spin column, then centrifuge at 8000rpm for 1 minute.
Discarded the collection tube containing the flow-through solution, then placed the column into a
new 2ml collection tube
 Added 500µl of wash buffer WB I (with ethanol added), then centrifuge 8000rpm for 1 minute.
Discarded the flow-through and place the column back into the collection tube.
 Added 500µl of wash buffer WB II (with ethanol added) to the column, then centrifuge at 14000
for 3 minutes.
 Empty the collection tube, then placed the purification column back into the tube. Centrifuge at
14000rpm for 1 minute.
 Discarded the collection tube containing the flow-through solution, then transferred the column to
a sterile 1.5ml microcentrifuge tube
 Elution of DNA in 100µl of elution buffer in two steps in the same tube. Added 50µl of elution
buffer to the center of the column membrane to eluted genomic DNA. Incubated for 4 minutes at
room temperature, then centrifuge at 1000rpm for 2 minutes. Added 50µl of elution buffer again in
the center of the column membrane, incubated for 4 minutes at room temperature then centrifuge
at 1000rpm for 2 minutes.
 Discarded the purification column and store at -20ċ.
Extraction of DNA from chorionic villi (CVS)
Chorionic villus sampling (CVS) is a prenatal test used to detect birth defects, genetic diseases, and other
problems during pregnancy. During the test, a small sample of cells (called chorionic villi) is taken from
the placenta where it attaches to the wall of the uterus. Chorionic villi are tiny parts of the placenta that are
formed from the fertilized egg, so they have the same genes as the baby. CVS can help identify such
chromosomal problems as Down syndrome or other genetic diseases such as cystic fibrosis, Tay-Sachs
disease, and sickle cell anemia. CVS is considered to be 98% accurate in the diagnosis of chromosomal
defects. The procedure also identifies the sex of the fetus, so it can identify disorders that are linked to one
sex (such as certain types of muscular dystrophy that occur most often in males). CVS may carry a risk of
miscarriage, because the procedure is done in early pregnancy. Infection may also occur. Due to this, 10
weeks is generally the earliest recommended time to perform this test.

Required material:
 Ultrasound machine,
 sterile gloves and gown,
 antiseptic solution,
 needle,
 specimen container,
 local anesthetic,
 guidance device,
 medical drape,
 bandage,
 medical waste disposal supplies.

CVS sample collection protocol:

 The medical professional prepare to collect a tiny piece of the placenta, which is called chorionic
villus. This helps to see if the baby is developing normally.
 With the help of ultrasound, the medical professional locates the placenta inside the mother's belly.
This is like a map to find where to collect the piece from.
 The medical professional cleans the mother's belly so that everything is nice and clean around
where the placenta is.
 A small, thin needle is gently inserted into the belly to reach the placenta. A tiny piece of the
placenta, like a little sample, is carefully taken.
 Once the sample is collected, the medical professional takes out the needle and makes sure
everything is okay.

CVS Extraction Protocol:

Required material:
 Eppendorf,
 Microcentrifuge tube,
 micropipette (10µl-1000µl), (20µl-200µl), (1µl-20µl),
  tissue paper,
 incubator,
 Vortex machine,
 Centrifuge machine,
 Thermo-mixer,
 Vacuum dryer,
 Spin Column,
 Refrigerator (4ċ).

Required chemicals:
 saline solution or normal saline,
 CVS Lysis buffer (solution A),
 proteinase K, SDS,
 solution C+D,
 chloroform,
 3M sodium acetate,
 isopropanol,
 70% ethanol,
 TBE buffer.

Procedure:
 Labeled two or three eppendorf tubes NS (normal saline).
 Added saline solution or normal saline into labeled eppendorf
 Took CVS tissue in eppendorf tubes
 Added 50µl CVS Lysis buffer to CVS tissue (solution A)
 Added 50µl of proteinase K
 Left the tubes for incubation at 60ċ for 2 hours.
 Incubated the tissues until the solution became clear.
 For proper degradation, the incubation time may be increased
 Added 500µl of solution C+D centrifuge at 3000 rpm for 5 minutes.
 Transferred the supernatant to a new tube: added 500µl of solution C+D and centrifuge at 1300
rpm for 3 minutes.
 Transferred the supernatant to a new tube; added 500µl of solution C+D centrifuge at 13000 rpm
for 3 minutes.
 Transferred the supernatant to a new tube.
 Added 250µl of solution D and centrifuge for 4 minutes at 13000 rpm.
 Transferred the aqueous phase to a clean dry tube.
 Did not discard any tube until the extraction was done and the DNA is visualized.
 Added 250µl of chloroform and centrifuge for 4 minutes at 130000 rpm.
 Separated the aqueous layer and precipitate the DNA by added 250µl of 3M Na acetate and 500µl
of isopropanol. (if DNA threads are visible, keep the tubes at a freezing temperature for 2 hours. If
threads are not visible, keep the tubes overnight).
 Centrifuge for 4 minutes at 13000 rpm.
 Discarded the supernatant with care.
 Added 350µl of 70% ethanol and spinned for 4 minutes at 13000 rpm.
 Discarded the ethanol and dried the pellet.
 Added 30µl of TBE buffer.
 Stored at 4ċ for further analysis (Gel Electrophoresis or PCR).
Gel Electrophoresis:
Gel electrophoresis is a technique used in molecular biology and biochemistry to separate DNA, RNA, or
proteins based on their size and charge. It involves placing the molecules in a gel matrix and applying an
electric field, which causes the molecules to migrate through the gel. Smaller molecules move faster and
travel farther, while larger ones remain closer to the starting point. This separation allows researchers to
analyze and study the different components in a sample. It's commonly used in DNA sequencing,
genotyping, and protein analysis.

Agarose Gel protocol:

Required Material:
 Pipette,
 Measuring cylinder,
 Oven, flask

Required Chemicals:
 Agarose,
 0.5x TBE buffer

10x TBE Buffer Recipe (FOR 171ML):

Took 20.7g Tris, 10.57g Boric acid, 1.28g EDTA was dissolved with the help of magnetic stirrer in 131ml
distilled water and adjusted the pH 6.8 by adding HCL then finalized the volume up to 171ml.

0.5x TBE Buffer Recipe:

Took 50ml 10x TBE buffer and dissolved in 950ml distilled water.

Procedure:
 Weighed one gram of Agarose into a 250ml glass bottle or conical flask.
 Add 100ml of 0.5x TBE and swirl to mix.
 Microwave for about 2minutes to dissolve Agarose.
 Particularly if using a microwave oven-make certain that the cap of the bottle is loose (Steam
pressure build-up can make the bottle explode).
 Watched the Agarose solution carefully as you heated it, and stirred frequently to avoid
superheating and a sudden (very messy) eruption of Agarose all over the microwave.
 After the Agarose was completely dissolved, left it for cool.
 Added 5µl ethidium bromide in the Agarose gel and slightly dissolved in it.

Gel Electrophoresis Protocol:

Required Material:
  Comb
 Gel casting tray
 Horizontal Gel electrophoresis chamber
 Transilluminator,
 DNA sample

Required Chemicals:
 Loading dye,
 0.5X TBE Buffer
LOADING DYE RECIPE:
25mg bromophenol blue, 4g sucrose was dissolved in 10ml water.

Procedure:
 Set the tray inserted the comb and double checked that it was correctly positioned.
 Poured the gel slowly into the tank. Pushed any bubbles away to the side using a disposable tip.
 Allowed it cooling to room temperature for 30 minutes.
 Carefully removed the comb by pulling straight up, and carefully pop the tray loose from the mold.
 Placed the Agarose gel into the electrophoresis chamber (wells facing the negative electrode) and
just covered the top of the gel with 0.5X TBE buffer.

Preparing the sample:

 Took 1µl of bromophenol dye and placed on the aluminum foil.
 Took 6µl of DNA sample and mixed with dye on the foil.
 Adjusted the pipette according to the amount.
 Loaded these samples into the wells of the submarine gel.
 Closed the tank.
 Running the Gel Connected the electrophoresis chamber to a power supply.
 The DNA had migrated towards the positive (red) electrode. (Caution: These power supplies do
have the potential to give you a nasty, perhaps fatal shock if certain circumstances arise, therefore,
always shut off the power supply and disconnected the gel chamber before reaching into the buffer).
 Kept the electrophoresis area dried and cleaned, and never leaved a live electrical lead or open
buffer chamber exposed for others to (unpleasantly) discover.
 Turned on the power supply at 120v for 30 minutes and run the gel until bromophenol blue (fastest
migrating) dye is 7/8 down the gel.
 Shut off the current, unplug the gel electrophoresis box, and transferred the gel into one of the
plastic trays for staining.
Visualizing the DNA in the Gel to detect the DNA molecules that were resolved by the Agarose gel
electrophoresis protocol, made use of ethidium bromide, a dye that has a high affinity for binding to nucleic
acids. One molecule of DNA can bound many molecules of ethidium bromide (ethidium bromide
intercalates between the bases), and because the bound ethidium bromide can be made to brightly fluoresce
upon exposure to ultraviolet light, extremely small quantities of DNA can be Visualize by technique (as
little as 10 Nano grams).
(Caution: Ethidium bromide is a potent mutagen and a possible carcinogen. Therefore: always wear gloves).
Or added a sufficient quantity of a 2Lg/ml solution of ethidium bromide in water to just cover for 20 min.
washed the gel in your gel. Gently rock the staining solution on a shaker platform running water.
Analysis:
 Took the gel into the darkroom, and placed on the trans-illuminator (a light box that emits
shortwave ultraviolet light).
 Turned on the UV for few seconds and quickly turned off the UV light.
 Took the photographed for visualizing.
 Never use the Trans-illuminator without (Caution: UV output of the trans-illuminator wearing the
UV goggles provided).
 Eyeglasses are not necessarily opaque to UV and other types goggles (designed for shattor-
resistance) are not appropriate.
 Trans-illuminator was connected to the computer so that it could be photographed.
 Capture the gel image using the gel doc. System software.
 Save the image for further analysis.
 Although the stained nucleic acid fluoresces reddish-orange, images are usually shown in black
and white.
 When handling ethidium bromide, and dispose of used solutions and contaminated material.
Polymerase Chain Reaction (PCR):
PCR is a common laboratory (in vitro) technique used to make many copies (millions to billions) of a
particular region of DNA. This DNA region can be anything the experimenter is interested in. For
Example, it might be a gene whose function a researcher wants to understand, or a genetic marker used by
forensic scientists to match crime scene DNA with suspects.
Typically, the goal of PCR is to make enough of the target DNA region that it can be analyzed or used in
some other way. For instance, DNA amplified by PCR may be sent for sequencing, visualized by gel
electrophoresis, or cloned into a plasmid for further experiments.

Thermocycler:
A thermocycler is a laboratory instrument used to amplify DNA & RNA samples via the Polymerase Chain
Reaction. The device has a thermal block with holes where tubes with the PCR reaction mixture can be
inserted. The thermocycler raises and lowers the temperature of the samples in a holding block in discrete,
pre-programmed steps, allowing for denaturation and re-annealing of samples with various reagents.

Components of PCR:
• DNA template:
The sample DNA (original DNA) that contains the target sequence.

• DNA Polymerase:
A type of enzyme that synthesizes new strands of DNA complementary to the target sequence. The
first and most commonly used of these enzymes is Taq DNA polymerase (from Thermus
aquaticus), whereas Pfu DNA polymerase (from Pyrococcus furious) is used widely because of its
higher fidelity when copying DNA.
Although these enzymes are subtly different, they both have two capabilities that make them
suitable for PCR:
1) They can generate new strands of DNA using a DNA template and primers,
2) They are heat resistant.
• Primers:
Short pieces of single-stranded DNA that are complementary to the target sequence. The
polymerase begins synthesizing new DNA from the end of the primer (DNA polymerase can’t
 initiate DNA synthesis without a primer). Typical primers are 18-30 bases in length, having 50-
60% GC composition and have a balanced distribution of G/C & A/T rich domains.

• dNTPs (deoxynucleotidetriphosphates):
Single units of the bases A, T, G, and C, which are essentially "building blocks" for new DNA
strands.

• Buffer Solution:
Maintain PH and ionic strength of the reaction solution suitable for the activity of the enzyme.

• Mg+ ions:
Mg+ ions are an essential cofactors for polymerase enzyme activity. Mg2+ in the PCR mixture
stabilizes dsDNA and rise the Tm.

PCR Mixture Preparation Protocol:

Required Materials:
 DNA template,
 Forward and Reverse primers,
 dNTPs (nucleotides),
 DNA polymerase,
 Reaction buffer,
 Sterile distilled water,
 PCR tubes,
 Pipettes and pipette tips,
 Spinner.

Procedure:
• Determined the number of reactions were performed and calculated the volumes of reagents
required for each reaction. The reaction mixture included:

Components Volume taken for 20µl Volume taken for 4

reactions
PCR buffer 2.0µl 8.0µl
MgCl2 1.5µl 6.0µl
dNTPs 2µl 8.0µl
Forward primer (20mM) 0.3µl 1.2µl
Reverse primer (20mM) 0.3µl 1.2µl
Taq. Polymerase (u) 0.2µl 8.0µl
Distilled water 11.7µl 46.8µl
Template DNA 2µl 8.0µl

• Adjusted the total volume to desired reaction volume (e.g., 20μL per reaction)
• In a sterile microcentrifuge tube, prepared a Master Mix by combining the calculated volumes of
dNTPs, Taq. Polymerase, MgCl2 and PCR buffer. Mixed these components thoroughly.
 • Added the calculated volumes of forward and reverse primers to the master mix, as well as the
DNA template. Mixed gently.
• Added sterile distilled water to bring the total reaction volume up to the desired level. Mixed again.
• Carefully pipetted the PCR mixture into individual PCR tubes. Used a fresh pipette tip for each
transfer to avoid cross-contamination.

PCR Machine Protocol:

• Opened the lid of the PCR machine and placed the PCR tubes into the block.
• Ensured the tubes are properly seated and closed the lid securely.
• Power on the PCR machine.
• Used the machine interface to set the thermal cycling parameters:
Initial Denaturation Annealing Extension Cycles Final Hold
Denaturation Extension
Temperature 95ċ 95ċ 65ċ 72ċ 35 72ċ 4ċ
Time 10minutes 45seconds 45seconds 1minute ----- 10minutes -----
• Started the PCR reaction using the machine’s interface, the machine were automatically cycle
through the specified temperatures and times.
• Allowed the machine to complete all cycles.
• When the PCR was completed, the machine were typically hold the reaction at a final extension
temperature for a short period to ensure all products are fully extended.
• Once all the cycles were completed, carefully opened the lid of the PCR machine and removed the
PCR tubes from the block.
• The amplified PCR products can be used immediately for downstream applications or stored at an
appropriate temperature.
• Analyzed the PCR products using Gel Electrophoresis.
Primer:
A primer is a short sequence of nucleotides that is essential for the initiation of DNA synthesis. It functions
as the basis for DNA replication or amplification in techniques such as PCR (Polymerase Chain Reaction).
Primers are designed to be complementary to particular regions of template DNA, allowing them to bind
and serve as a starting point for DNA synthesis.
Typically, a pair of primers consisting of a forward primer and a reverse primer is used in DNA
amplification techniques. The forward primer is designed to run in the 3′-5′ direction, whereas the reverse
primer is designed to run in the 5′-3′ direction. Together, they bind to opposite extremities of the target
sequence, with the 3′ end of the forward primer corresponding to the DNA elongation template strand.
During DNA elongation, the primers direct the DNA polymerase to produce two new strands of double-
stranded DNA. The forward primer guides the synthesis of the new strand in the 5′-3′ direction, whereas
the reverse primer guides the synthesis of the complementary strand.
Nonetheless, the formation of dimers is a potential issue that may arise. When the template strand binds
with its complementary strand, an unfavorable condition results in the formation of dimers. In the design
of primers, it is essential to avoid such dimers.
Compared to RNA primers, DNA primers are more commonly used in polymerase chain reaction (PCR).
DNA primers are able to withstand the high temperatures involved in the denaturation phase of PCR
without degrading. Unlike RNA primers, DNA primers can be readily synthesized and used in the
unidirectional mode of polymerization. Primarily DNA primers are utilized in PCR for the amplification
of DNA.
During the replication process, primers have the ability to eliminate RNA primers at the conclusion.
However, DNA primers are not eliminated after DNA synthesis or amplification has been completed.
Therefore, it is essential to utilize nucleotide primers that target the desired region precisely.
Types of Primers:
There are two main types of primers: DNA primers and RNA primers. DNA primers are commonly used
in vitro, specifically for PCR amplification and DNA sequencing. On the other hand, RNA primers are
utilized in in vivo processes like DNA replication and cloning.
1. DNA Primers:
 Application: Used in vitro for PCR amplification and DNA sequencing.
 Reaction: The amplification process is temperature-dependent and requires fewer proteins.
 Length: Typically 18-24 base pairs.
 Synthesis: DNA primers are chemically synthesized.
 Stability: DNA primers are long-lived and more stable.
2. RNA Primers:
 Application: Primarily used in in vivo processes like DNA replication and cloning.
 Reaction: The replication process is a catalytic reaction that relies on enzymes and involves several
proteins.
 Length: Generally 10-20 base pairs.
 Synthesis: RNA primers are synthesized using the Primase enzyme.
 Stability: RNA primers are short-lived and more reactive.

During DNA replication, DNA polymerase initiates the addition of nucleotides to the reactive 3′ (OH) end
of the existing nucleic acid, facilitating the elongation and replication of the parent strand. DNA primers
are preferred in various applications due to their stability, ease of storage, and the requirement of fewer
enzymes to initiate synthesis.
The choice between DNA primers and RNA primers depends on the specific experimental requirements
and the nature of the process, whether it is in vitro amplification or in vivo replication and cloning. DNA
primers are favored for their longevity and stability, while RNA primers are more short-lived and reactive.

Primer Designing:
Primer designing is a critical process that involves considering several essential factors. Let’s explore these
factors and their significance:

• Primer Length:
Optimal primer length is typically between 18 to 24 nucleotides. Shorter primers facilitate easy
binding to the template at the annealing temperature during PCR.
• Melting Temperature (Tm):
Tm refers to the temperature at which half of the primer-template duplexes are dissociated. The GC
content of the primer sequence provides an estimate of Tm. The difference in Tm between the
forward and reverse primers should not be less than 2°C.
• Primer Annealing (Ta):
Ta is the temperature at which primers anneal to the template DNA during PCR. A high Ta can
result in insufficient primer-template hybridization and low PCR product yield. Conversely, a low
Ta may lead to non-specific PCR products due to a high number of mismatches. Ta can be
calculated using the equation: Ta = 0.3 * Tm (primer) + 0.7 * Tm (product) – 14.9.
• Primer GC Content and Clamp:
For gene sequencing, primers should ideally have a GC content between 40% and 60%. A GC
clamp, consisting of two GC bases at the 3′ end of the primer, enhances primer stability and
improves binding specificity. GC base pairs form three hydrogen bonds, which are stronger than
the two hydrogen bonds in AT base pairs, contributing to primer stability.
• Setting Restriction Enzyme (RE) Cut Sites:
When designing primers for cloning or restriction enzyme digestion, adding 3-5 bases to the 5′ end
of the target primer can create a leader sequence that facilitates efficient cutting by the
corresponding restriction enzyme.
• End Stability:
A stable 3′ end of the primer, often achieved by incorporating a maximum number of G bases,
improves primer binding specificity and reduces the chances of false priming.

By considering these factors, researchers can design primers that are optimized for specific applications,
such as PCR amplification, gene sequencing, or cloning, leading to successful and specific amplification of
the target DNA sequence.

Designing PCR Primers Cautions

When designing PCR primers, certain cautionary aspects need to be considered. Here are some cautionary
factors and their significance:

• Hairpins:
 Hairpin structures are formed by intramolecular interactions within a primer, specifically at the 3′
end. It is generally acceptable if the hairpin structure has a stability of -2 kcal/m, and internal
hairpins should have a stability of -3 kcal/m.

• Repeats and Runs:

The presence of consecutive dinucleotide repeats or runs within a primer can affect its specificity
and efficiency. It is important to limit the occurrence of repeats and runs, with a maximum of 4
dinucleotide and 4 base pairs.

• Dimers:
Dimers are formed when two primers interact with each other, resulting in double-stranded DNA.
Self-dimers occur when two identical or homologous primers interact, while cross-dimers refer to
interactions between forward and reverse primers. Care should be taken to avoid both self-dimer
and cross-dimer formation.

• Primer-Template Cross Homology:

Primers should be designed to have minimal homology within the template sequence, except for
the target site. Intra-primer homology refers to complementary bases within the same primer, with
regions longer than 3 bases potentially causing intramolecular bonding. Inter-primer homology
occurs when forward and reverse primers have complementary sequences, leading to
intermolecular bonding.
• Primer Dimer Formation:
It is crucial to analyze and prevent the formation of primer dimers. Primer dimer formation can be
assessed by calculating the Gibbs free energy. Analyzing the 5′ end of the primer has been found
to be more reliable than the 3′ end.

Considering these cautionary factors helps ensure the specificity, efficiency, and accuracy of PCR
amplification. By avoiding hairpins, dimers, excessive repeats and runs, unintended homology, and primer
dimer formation, researchers can design primers that specifically target the desired DNA sequence and
minimize non-specific binding and amplification.

Primer Designing Tools:

 Ensembl
Ensembl is a comprehensive and user-friendly genome browser and data resource that offers a
wealth of genomic information for a wide range of species. It allows researchers, scientists, and
bio-informaticians to explore, analyze, and visualize genomic data, gene annotations, regulatory
elements, genetic variations, and more. Ensembl's interactive interface and extensive database
make it a valuable tool for understanding the structure, function, and evolution of genomes, aiding
various biological and genetic research endeavors.
 Primer 3
Primer3 is a widely used computer program designed for designing polymerase chain reaction
(PCR) primers. It assists researchers in selecting optimal primers for amplifying specific DNA
 sequences during PCR experiments. The program takes into consideration various factors like
primer length, melting temperature, and potential secondary structures to generate primers that
enhance the efficiency and specificity of the PCR process.
 Primer Blast
Primer-BLAST is a tool provided by the National Center for Biotechnology Information (NCBI)
that helps design PCR primers for specific DNA sequences while also checking for potential off-
target amplification. It combines the functionality of primer design with a sequence similarity
search to ensure that the selected primers will specifically amplify the intended target sequence and
not cross-amplify similar sequences in the genome. This helps researchers achieve more accurate
and specific PCR results.
 NCBI
NCBI stands for the National Center for Biotechnology Information. It is a part of the United States
National Library of Medicine, which is a branch of the National Institutes of Health (NIH). NCBI
provides various databases, tools, and resources related to molecular biology, genetics, and
biotechnology, including resources like Gen-Bank, PubMed, and BLAST. It's a valuable hub for
researchers and scientists in the life sciences field.
 Oligo Calculator
“Oligo Calculator" typically refers to a web-based tool or software used by molecular biologists
and researchers to calculate parameters for designing and working with oligonucleotides.
Oligonucleotides are short sequences of nucleotides (DNA or RNA) used in various molecular
biology experiments, such as PCR, sequencing, and gene synthesis. OligoCalculator helps
researchers determine parameters like melting temperature (Tm), molecular weight, and other
characteristics that are crucial for designing successful experiments involving oligonucleotides. It
aids in ensuring the specificity and efficiency of these experiments.
 Oligo Analysis Tool
An "oligoanalysis tool" is a software or online resource designed to analyze and assess
various characteristics of oligonucleotides. Oligonucleotides are short sequences of
nucleotides, typically DNA or RNA, used in molecular biology applications. An
oligoanalysis tool helps researchers and scientists evaluate important parameters of
oligonucleotides, such as melting temperature (Tm), secondary structure formation,
potential primer-dimer interactions, and other factors that can impact their performance in
experiments like PCR, sequencing, and gene expression analysis. These tools contribute to
the effective design and optimization of oligonucleotide-based experiments.
 Bio-web
"The Bio-Web: Resources for Molecular and Cell Biologists" is a non-commercial, educational site
with the only purpose of facilitating access to biology-related information over the internet.
 Insilico PCR (UCSC)
The term "Insilico PCR" refers to a computational method used to simulate or predict the
Polymerase Chain Reaction (PCR) amplification of DNA sequences in silico, meaning in a
computer or using software. This technique allows researchers to virtually assess the potential
success of PCR experiments without physically conducting the reactions in the laboratory. It's often
used for primer design, optimizing reaction conditions, and predicting amplification outcomes.
1) Primer designing through Ensembl

 Go to ensembl to acquire sequence

 Identify target sequence (use 5’ flanking sequence for forward primer and 3’ flanking sequence for
reverse primer)

 Copy the desire sequence

 Paste on MS word
 Go to Oligocalculator

 Paste few base pair randomly selected from desire sequence intron from 3’ and from 5’
 To check the self-complementary of manually selected primer
 Go to Bio web
 Go to sequence cleaner
 Paste the reverse primer to check the reverse complementary
 Go to UCSC
 Click Insilico PCR

 To check the primer that they were successful

2)Primer designing through primer 3
 Go to NCBI

 Click the gene option

 Select the gene
 Click FASTA
 Copy desired sequence
 Past on MS word
 Select random sequence from MS word
 Go to bio web
 Clean the sequence
 Go to primer 3

 Paste the random sequence and checked suitable primer

 Select the primer that were suitable for all parameters (dimer formation, self-complementary,
hairpin structure, primer-primer interaction etc.)
 For forward primer go to MS word and paste the primer in navigation to match the primer to the
sequence
 For reverse primer go to bio web to check reverse complement
 Select the primer go to MS word and paste the primer in navigation to match the primer to the
sequence.
 Go to oligoanalysis Tool
3)Primer designing through primer blast
 Go to NCBI

 To acquire sequence
 Go to FASTA
 Copy desire sequence and paste on MS word
 Go to primer blast

 Paste the whole selected sequence

 Go to again MS word and count the word for forward primer and reverse primer and set all
parameters in primer blast
 Wait for few minutes
 Detail primer report shown but select the one which is suitable for desire gene
 Copy the primer and paste on MS word
 Go to UCSC
 Click Insilico PCR to check sequence
 To check binding sites of forward and reverse primer go to MS word and paste the sequence in
navigation
 Go to Bio web
 Clean the sequence
 Go to Oligo analysis Tool
Pedigree
A pedigree is a graphical representation of a family's genetic history that shows the relationships between
individuals and indicates the presence or absence of specific traits or conditions within that family. It's often
used in genetics to study inheritance patterns and trace the transmission of genetic traits or diseases across
generations.

Reading a Pedigree

Pedigrees represent family members and relationships using standardized symbols.

By analyzing a pedigree, we can determine genotypes, identify phenotypes, and predict how a trait will be
passed on in the future. The information from a pedigree makes it possible to determine how certain alleles
are inherited: whether they are dominant, recessive, autosomal, or sex-linked.

To start reading a pedigree:

 Determine whether the trait is dominant or recessive

 If the trait is dominant, one of the parents must have the trait. Dominant traits will not skip a
generation. If the trait is recessive, neither parent is required to have the trait since they can be
heterozygous.

 Determine if the chart shows an autosomal or sex-linked (usually X-

linked) trait
For example, in X-linked recessive traits, males are much more commonly affected than females.
In autosomal traits, both males and females are equally likely to be affected (usually in equal
proportions).

Example: Autosomal Dominant Trait

The diagram shows the inheritance of freckles in a family. The allele for freckles (F) is dominant to the
allele for no freckles (f).

At the top of the pedigree is a grandmother (individual I-2) who has freckles. Two of her three children
have the trait (individuals II-3 and II-5) and three of her grandchildren have the trait (individuals III-3, III-
4, and III-5).

Example: X-linked recessive trait

The diagram shows the inheritance of colorblindness in a family. Colorblindness is a recessive and X-linked
trait (Xb). The allele for normal vision is dominant and is represented by (XB).
In generation I, neither parent has the trait, but one of their children (II-3) is colorblind. Because there are
unaffected parents that have affected offspring, it can be assumed that the trait is recessive. In addition, the
trait appears to affect males more than females (in this case, exclusively males are affected), suggesting that
the trait may be X-linked.

HaploPainter (A tool to draw pedigree)

HaploPainter is a specialized software tool designed for creating and visualizing pedigrees, particularly in
the context of genetic and hereditary studies.

Purpose:
HaploPainter is primarily used by geneticists, researchers, and clinicians to create detailed and informative
pedigree charts. These charts help in analyzing and understanding the inheritance of genetic traits, diseases,
and markers within families.

Features:
 Pedigree Drawing:
HaploPainter allows users to draw pedigrees, representing family relationships and generations.
 Genetic Markers:
Users can include genetic markers, genotypes, and haplotypes on the pedigree to study inheritance
patterns.
 Customization:
The tool offers customization options for colors, shapes, and labels, making it easier to differentiate
between various family members and traits.
 Visualization:
HaploPainter provides clear visualizations of complex genetic data, aiding in the interpretation of
inheritance patterns.

Data Input:
Users can input genetic data into HaploPainter in various formats, such as linkage and haplotype data,
genotypes, or directly from genetic database files.

Output:
The tool generates detailed and informative pedigree charts that can be saved or exported for presentations,
research papers, or clinical discussions.
HaploPainter is often an open-source tool, which means it’s freely available to the scientific community.
CYTOGENETICS/KARYOTYPING LAB
Karyotyping
Karyotyping is a laboratory technique used to examine the chromosomes of an individual. It helps identify
and analyze the number, size, and shape of chromosomes in a cell sample. Karyotype also called Idiogram.
We obtain chromosomes in metaphase, in which chromosomes are best visualized. By arranging and
matching the chromosomes, it can detect abnormalities, such as chromosomal disorders like Down
syndrome. This technique is commonly used in genetic testing and research to understand genetic variations
and diagnose certain genetic conditions.

Blood Collection Protocol:

Required Material:

 Syringe,
 tourniquet,
 blood collection tubes or vial,
 Anticoagulant,
 Alcohol or disinfectant,
 holder/Adaptor,
 Gloves,
 Gauze sponge

Procedure:
 First explained the procedure to the patient and obtained their consent.
 Identified a suitable site for blood collection, usually the inner elbow area or the back of the hand
or peripheral vein.
 Cleaned the skin at the selected site with alcohol or a disinfectant and allowed it to dry.
 Used a needle and heparinized syringe or a vacutainer system to draw the required amount of blood
from the vein. The amount of blood collected depends on the karyotyping protocol and the intended
downstream applications.
 Mixed the collected blood with the appropriate anticoagulant (e.g., lithium heparin) to prevent
clotting and prevent cell damaging.
 Properly labeled the blood collection tube with the patient's details and any relevant information.

Cell Culture Protocol:

Required Material:

 RPMI-1460 Medium,
 blood sample,
 Tissue culture flask,
  pipette,
 syringe,
 laminar air flow chamber,
 PHA,
 antibiotic (penicillin/ ampicillin/ streptomycin),
 Incubator.

Procedure:
 Firstly sterilized laminar air flow with spray of alcohol.
 Labeled the tissue culture flask with patient name, sample collected date and time.
 Sterilized the media with flame.
 Poured 10ml media in flask and then sterilized flask with flame to prevent contamination.
 Took 1cc blood sample in syringe and then slowly poured into the flask.
 Gradually added 80µl or 40 drops of PHA in flask.
 Added desire antibiotic to prevent microbial growth.
 Incubated at 37ċ for 72 hours.

Harvesting Protocol:
Required Material:

 Colchicine,
 centrifuge machine,
 falcon tubes,
 incubator,
 pipette,
 hypotonic solution,
 Fixative.

Solutions Preparation:

 Hypotonic Solution Recipe:

Took 0.42g KCl and added 75ml of distilled water and mixed well.

 Fixative Recipe:
Took 10ml Glacial acetic acid in 30ml methanol and made the ratio of 1:3.

Procedure:
 After 72 hours took sample from incubator and then added 22µl colchicine in each flask to arrest
cell division at the metaphase stage, where chromosomes are condensed and visible.
 Placed flask at room temperature for 30 minutes.
 After 30 minutes poured sample in falcon tube and then centrifuge at 4000rpm for 10 minutes.
  Discarded supernatant.
 Added 10ml hypotonic solution in each flacon tube and then incubated at 37ċ for 20 minutes.
 After 20 minutes added 500µl fixative drop by drop in each tube to preserve the arrested cells and
their chromosomes in a spread-out state.
 Mixed them until it shows black color to ensured purity of culture.
 Centrifuge at 4000rpm for 10 minutes.
 Discarded supernatant and then washed with 7-8ml fixative and then centrifuge at 4000rpm for 10
minutes.
 Discarded supernatant and then repeated this process 2-3 times until it became white in color.
 Stored at -4ċ for further process.

Staining Protocol:
Required Material:

 Slides,
 Ethanol + HCl,
 tissue paper,
 fixative,
 centrifuge machine,
 phosphate buffer,
 distilled water,
 hot plate magnetic stirrer,
 incubator,
 slide container,
 vacuum dryer,
 staining solution,
 slides rack,
 falcon tubes,
 incubator,
 pipette.

Solutions Preparation:

 PHOSPHATE BUFFER RECIPE:

3.4g phosphate was dissolved in 1liter of distilled water adjusted pH at 6.8.

 GIEMSA DYE RECIPE:

Mixed glycerol and giemsa then left for 4 days for continuous stirring.

 STAINING RECIPE:
Took 13ml from phosphate buffer, 5ml methanol, 20µl giemsa dye ,420µl from trypsin mixed in a tube .
Procedure:
 Labelled slides with sample number/name.
 Then cleaned the slides with ethanol and HCL wiped it with tissue paper.
 Washed the slides with distilled water so that slides were neutralized after dry.
 Again washed the sample with fixative and centrifuge at 4000rpm for 10 minutes.
 Discarded the supernatant but left equal amount of supernatant to pellet mass then resuspended the
vials.
 After added 40µl of sample on slide placed it on hot plate stirrer for heat shock so that cells were
denatured (avoided over heating the slide).
 Then placed the slides in incubator for 90ċ for denatured extra protein except heterochromatin
protein for 1 hour or 54ċ for overnight.
 After incubation waited for 10 minutes to proceed next step.
 Placed the slides in slide container that had phosphate buffer then incubated the slide container at
56ċ for 13 minutes.
 Then washed slides with distilled water.
 Dried the slides with vacuum dryer.
 Then placed the slides on slide rack for stained.
 Poured 3ml staining solution on each slides with the help of pipette.
 Placed the slides on darker environment for 15 minutes.
 Washed the slides with distilled water and then air dried.
 Slides were ready for screening analysis under microscope.

Screening Protocol:
Procedure:
 Examine the stained chromosomes under CV Chromoscan device to identify and photograph them.
 Took the slide under specimen of microscope, microscope had attached with camera which capture
the photograph of disperse chromosome systematically, showed in connected Computer.
 Made new case file in cytodivision.
 Clicked on capture screen, then software capture photograph automatically.
 Added oil in slide which made metaphase more clearly in 100X.
 After found clear shape of chromosome where less overlapping, capture the photo
 Pressed capture setup-bright field, then red appearance given distribution of chromosome
 Clicked analysis, then clicked on split option so that separate the chromosome.
 Clicked draw arcs and selected the chromosome, this option suitable when more complex
overlapping seems in chromosome.
 Clicked auto classify so that it localized the chromosome.
 After found chromosome, Arrange the photographed chromosomes in pairs according to their size,
shape, and banding patterns.
 Created a karyogram, which was a visual representation of the individual's chromosome pairs,
typically displayed in a standardized format then saved the image and printed it
 If any error occurred repeat it again.
INDUSTRIAL BIOTECHNOLOGY DIVISION
Synthesis of Nano Particles Lab-1
Demonstration of Nano Particle Lab
Introduction:
The first scientific description of nanoparticle properties was provided by Michael Faraday in his famous
paper “Experimental Relations of Gold and Other Metals to Light”. The term “nanotechnology” was used
for the first time by Richard Feynman in 1959, which is considered to mark the beginning of modern
nanotechnology. The preface nano comes from a Latin nanos meaning “dwarf” that means extremely small.
Consequently, the nano-materials are usually dignified in nano-meters (1nm is equivalent to 10−9 m) and it
comprises systems having size less than macroscopic measurements and greater than molecules ones
(mostly >1nm and 100nm) at least in one spatial dimension. These nano-structures are made from basic
units or blocks having small dimensionality i.e. zero, one, two and three dimensions. In zero dimensional
nano-particles, the moment of electrons is cramped in all three dimensions, e.g. quantum dots. If electrons
can move freely in x-direction only, they are one dimensional nanoparticles e.g. quantum wires. Whereas,
in two dimensional thin films and three dimensional nano-structured materials, free electrons can move
freely in x, y and x, y, z directions respectively.
Note: Their greater surface are often makes them more sensitive, explosive and reactive.

Definition:
Nanotechnology can be defined as manipulation of materials at the atomic level by a combination of
engineering, chemical, and biological approaches.

Preparation of nano-particles:
The nano-particles can be prepared by various processes divided into i.e. bottom up and top down
techniques. Bottom up methods include the reduction of material components up to the atomic level and
then with further self-assembly lead to the formation of nano-particles. However, during self-assembly, the
physical forces functioning at nano-scale are used to connect basic units into macro structures. Pyrolysis,
bio-synthesis, sole gel, spinning and chemical vapor deposition are most extensively used methods fall in
this approach. Whereas, top down techniques including sputtering, laser ablation, nano-lithography,
mechanical milling and thermal decomposition, starting with a pattern produced on a higher scale, then
compacted to nanoscale.
 Different approaches and methods for synthesizing nanoparticles

Nanoparticles can be synthesized using a variety of methods including physical, chemical, biological, and
hybrid techniques. The production of nanoparticles through conventional physical and chemical methods
results in toxic by products that are environmental hazards. Additionally, these particles cannot be used in
medicine due to health-related issues, especially in clinical fields. Conventional methods can be used to
produce nanoparticles in large quantities with defined sizes and shapes in a shorter period of time; however,
these techniques are complicated, costly, inefficient, and outdated. In recent years, there has been growing
interest in the synthesis of environmentally friendly nanoparticles that do not produce toxic waste products
during the manufacturing process. This can only be achieved through benign synthesis procedures of a
biological nature using biotechnological tools that are considered safe and ecologically sound for
nanomaterial fabrication as an alternative to conventional physical and chemical methods [5]. This has
given rise to the concept of green technology or green nanobiotechnology. In general, green
nanobiotechnology means synthesizing nanoparticles or nanomaterials using biological routes such as those
involving microorganisms, plants, and viruses or their byproducts, such as proteins and lipids, with the help
of various biotechnological tools. Nanoparticles produced by green technology are far superior to those
manufactured with physical and chemical methods based on several aspects. For example, green techniques
eliminate the use of expensive chemicals, consume less energy, and generate environmentally benign
products and byproducts.

Factors Affecting the Synthesis of Green Nanoparticles:

There are several factors that affect the synthesis, characterization, and application of nanoparticles. Many
researchers have reported the change in nature of the synthesized nanoparticles with the type of the adsorbate and
the activity of the catalysts used in the synthesis process [18, 19]. Some of them have reported the dynamic nature of
the synthesized nanoparticles with different types of symptoms and implications by change with time and
environment, and so forth. Some dominant factors that affect nanoparticle biosynthesis are described below:

 Particular Method or Technique:

There are different methods for synthesizing nanoparticles, ranging from physical techniques using mechanical
procedures to chemical or biological protocols using various organic or inorganic chemicals and living organisms.
Each procedure has specific benefits and drawbacks. However, biological methods for synthesis of nanoparticles use
nontoxic and environmentally benign materials in conjunction with green technology and are therefore eco-friendly
and more acceptable than traditional methods.
 Temperature:
Temperature is another important parameter that affects the synthesis of nanoparticles using all three methods. The
physical method requires the highest temperature (>350∘C), whereas chemical methods require a temperature less than
350∘C. In most cases, the synthesis of nanoparticles using green technology requires temperatures less than 100∘C or
ambient temperature. The temperature of the reaction medium determines the nature of the nanoparticle formed.
 Pressure:
Pressure is important for the synthesis of nanoparticles. The pressure applied to the reaction medium affects the shape
and size of the synthesized nanoparticles. The rate of reduction of metal ions using biological agents has been found
to be much faster at ambient pressure conditions.
 Time:
The quality and type of nanoparticle synthesized using green technology are greatly influenced by length of time for
which the reaction medium is incubated.

 Preparation Cost:
To facilitate the potential application of nanoparticles in modern day uses, the costs associated with their
synthesis need to be regulated and controlled. Thus, the cost-effectiveness of the production procedure is
an important factor that influences nanoparticle synthesis
 Particle Shape and Size:
Particle size plays an important role in determining the properties of nanoparticles. For example, the
melting point of nanoparticles has been reported to decrease when the size of the nanoparticles reached the
nanometer scale. Nanoparticles with different configurations have similar energy that makes the
transformation of their shape easy. The type of energy commonly used during the analysis of the
nanoparticles stimulates the change in the shape of the nanoparticle. The dynamic nature and shape of the
synthesized nanoparticles greatly affect their chemical properties.
  Pore Size:
The quality and application of nanoparticles are greatly influenced by the porosity of the synthesized
nanoparticles. Immobilization of biomolecules onto nanoparticles has been achieved to increase their use
in the drug delivery and biomedical field.
 Proximity:
When the individual or isolated nanoparticles come in contact or near to the surface of other nanoparticles,
alteration in their properties is observed in most of the cases. This changing behavior of the nanoparticles
can be utilized in making more tuned nanoparticles. There are many implications of the proximity effect of
nanoparticles such as the particle charging, the substrate interactions, and magnetic properties of the
nanoparticles.
Characterization of the Synthesized Nanoparticles:
The nanoparticles present a range of characterization challenges that affect the detailed and appropriate
characterization of nanoparticles. Thus understanding the problems faced during characterization of
nanoparticles and selecting a suitable characterization technique are of utmost importance. Specifically,
nanoparticle characterization is performed to assess the surface area and porosity, pore size, solubility,
particle size distribution, aggregation, hydrated surface analysis, zeta potential, wettability, adsorption
potential and shape, size of the interactive surface, crystallinity, fractal dimensions, orientation, and the
intercalation and dispersion of nanoparticles and nanotubes in nanocomposite materials. Several techniques
can be used to determine nanoparticle parameters, including ultraviolet- (UV-) visible spectroscopy, atomic
force microscopy (AFM), transmission electron microscopy (TEM), scanning electron microscopy (SEM),
dynamic light scattering (DLS), X-ray photoelectron spectroscopy (XPS), thermogravimetric analysis
(TGA), powder X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR),matrix-assisted
laser desorption/ionization time-of-flightmass spectrometry (MALDI-TOF), dual polarization
interferometry, nuclear magnetic resonance (NMR), nanoparticle tracking analysis (NTA) for evaluation of
Brownian motion, and particle size analysis. Some of the important characterization techniques designed
to measure specific parameters are described in detail below:
 Ultraviolet-visible spectrophotometry:
Provide information regarding the size, stabilization, and aggregation of nanoparticles.

 Transmission electron microscopy:

Determine the shape, size (10-exp10m), morphology and allographic structure of the nanoparticles.

 High-resolution transmission electron microscopy:

Determine the arrangement of the atoms and their local microstructures, such as lattice fringe, glide
plane, lattice vacancies and defects, screw axes, and surface atomic arrangement of crystalline
nanoparticles.
 Scanning electron microscopy:
Determine the morphology by direct visualization.
  Atomic force microscopy:
Determine the size information (length, width, and height) and other physical properties (such as
morphology and surface texture)
 Dynamic light scattering:
Determine the particle size distribution.
 Zeta potential:
Determine the stability and surface charge of the colloidal nanoparticle, as well as nature of the
materials encapsulated inside the nanoparticle or coated on ots surface.
 Fourier transform infrared spectroscopy:
Characterize the nanoparticles to understand their functional groups and determine the emission,
absorption, photoconductivity, or Raman scattering of a solid, liquid, or gas.
 X-ray photoelectron spectroscopy:
Determine the mechanism of the reaction that occurs on the surface of magnetic nanoparticles and
other characteristics involved in the bonding of different elements involved, as well as confirming
the structure and speciation of different elements present in the chemical composition of the
magnetic nanoparticles.
 Thermal gravimetric analysis:
Confirm the formation of coatings such as surfactants or polymers to estimate
the binding efficiency on the surface of magnetic nanoparticles
 Chromatography and relative techniques:
Separate nanoparticles on the basis of their affinity towards the mobile phase
 Electro spinning technique:
Electro spinning involves an electro hydrodynamic process, during which a liquid droplet is
electrified to generate a jet, followed by stretching and elongation to generate fiber.
Green synthesis of nanoparticles:
Green synthesis of silver nanoparticles makes use of plant constituents, like carbohydrates, fats, enzymes,
flavonoids, terpenoids, polyphenols, and alkaloids, as reducing agents to synthesize silver nanoparticles.
The present study for the first time utilized seed extract of Tectona grandis (teak) for reduction of 1 mM
silver nitrate solution to silver nanoparticles. The method proved to be very simple, cost-efficient, and
convenient. Synthesis of nanoparticles was confirmed by visual detection in which the colorless solution
gets changed to a brown-colored solution. Further characterization was done by UV-visible spectroscopy,
XRD, FTIR analysis, SEM/EDS, FESEM, and TEM. Size of silver nanoparticles was found to be 10–30
nm approximately as determined by transmission electron microscopy (TEM). Energy-dispersive spectra
(EDS) revealed that nanoparticles contain silver in its pure form. Well diffusion method showed the
antimicrobial effect of AgNPs on different microorganisms with the zone of inhibition of 16 mm for
Staphylococcus aureus, 12 mm for Bacillus cereus, and 17 mm for E. coli when 50 μg of AgNPs was used.
Minimum inhibitory concentration was found to be 5.2, 2.6, and 2.0 μg/ml for Bacillus cereus,
Staphylococcus aureus, and E. coli respectively. Mode of action of antimicrobial activity of nanoparticles
was investigated by determining leakage of reducing sugars and proteins, suggesting that AgNPs were able
to destroy membrane permeability.
Method:
Tectona grandis seeds were collected from BHU campus. Before washing, the outer covering of the
harvested seeds was removed. Seeds were dried at room temperature for 3–4 days so that moisture gets
removed completely. Dried seeds were crushed to fine powder and stored in dry and airtight container for
further use. Silver nitrate (AgNO3) was purchased from SRL, India. All other reagents were of analytical
grade and used as received. All the solutions were freshly prepared with double distilled water for the
experimental procedure and were kept in the dark to avoid any photochemical reaction.
Tectona grandis seed extract preparation:
Five grams of seed powder was carefully weighed and added in an Erlenmeyer flask of 250 ml containing
50 ml of double distilled water. The mixture in the flask was heated in a water bath for 15–20 min at 80 °C.
After boiling, the extract was filtered using a muslin cloth to remove any coarse material. Volume was
made up to 100 ml using double distilled water. The prepared extract was stored at 4 °C and used within 1
week.
Synthesis of silver nanoparticles using seed extract:
1 mM silver nitrate solution in double distilled water was the source of silver. Silver nitrate and seed extract
were mixed together in a ratio of 1:9. The reaction mixture was heated below the boiling point and
continuously stirred at 800 rpm using magnetic stirrer. The mixture turned reddish brown in color within 1
h. The whole reaction was carried out in the dark. The obtained suspension of Ag/T. grandis was centrifuged
at 15,000 rpm for 45 min. The pellet containing silver nanoparticles was washed 3–4 times with deionized
water to remove silver ions and seed extract residue. The precipitated nano-particle were lyophilized.
Lyophilized nanoparticles were stored in a cool, dry, and dark place and further their characterization was
carried out.

Antibacterial assay:
Well diffusion method is commonly used to check the antimicrobial activity of the nanoparticles. The
antimicrobial activity of synthesized AgNPs was checked using this method. Three microorganisms,
namely E. coli, Bacillus cereus, and Staphylococcus aureus were used for this purpose. 1 mg/ml solution
of AgNPs was made in Milli Q water and was sonicated properly. Overnight grown culture in Luria-Bertani
broth of the mentioned microorganisms was taken and diluted to an optical density of 1. Diluted bacterial
suspension (500μl) was spread uniformly on three different Luria-Bertani agar plates for three different
microorganisms. After spreading, two wells were cut on each plate using well borer of approximately 10
mm diameter. One well was filled with 50 μg and the other with 100μg of AgNPs solution in all the three
plates. The plates were incubated at 37 °C for 24 h, and the zone of inhibition was measured.
Biotechnology of Pre-Biotic & Anti-Microbial Lab:
Introduction:
The prebiotics concept was introduced for the first time in 1995 by Glenn Gibson and Marcel Roberfroid.
Various types of microorganisms, known as gut microbiota, are inhabitants of the human gastrointestinal
tract. It has been reported that there are 10exp10–10exp12 live microorganisms per gram in the human
colon. The resident microbial groups in the stomach, small, and large intestine are crucial for human health.
The majority of these microorganisms, which are mostly anaerobes, live in the large intestine. Although
some endogenous factors, such as mucin secretions, can affect the microbial balance, human diet is the
chief source of energy for their growth. Particularly, non-digestible carbohydrates can highly modify the
composition and function of gut microbiota. Beneficial intestinal microbes ferment these non-digestible
dietary substances called prebiotics and obtain their survival energy from degrading indigestible binds of
prebiotics. As a result of this, prebiotics can selectively influence gut microbiota.
On the other hand, the gut microbiota affects intestinal functions, such as metabolism and integrity of the
intestine. Moreover, they can suppress pathogens in healthy individuals through induction of some
immunomodulatory molecules with antagonistic effects against pathogens by lactic acid that is produced
by Bifidobacterium and Lactobacillus genera. Various compounds have been tested to determine their
function as prebiotics. Fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), and trans-galacto-
oligosaccharides (TOS) are the most common prebiotics. Fermentation of prebiotics by gut microbiota
produces short-chain fatty acids (SCFAs), including lactic acid, butyric acid, and propionic acid. These
products can have multiple effects on the body. As an example, propionate affects T helper 2 in the airways
and macrophages, as well as dendritic cells in the bone marrows. SCFAs decrease the pH of colon.
Peptidoglycan is another prebiotics fermentation product that can stimulate the innate immune system
against pathogenic microorganisms. The structure of prebiotics and the bacterial composition of gut
determine the fermentation products. The effects of prebiotics on human health are mediated through their
degradation products by microorganisms. For example, butyrate influences intestinal epithelial
development. Since SCFAs can diffuse to blood circulation through enterocytes, prebiotics have the ability
to affect not only the gastrointestinal tract but also distant site organs.

Definitions:
Prebiotic was described as “a non-digestible food ingredient that beneficially affects the host by selectively
stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improves
host health”.
This definition was almost unchanged for more than 15 years. According to this definition, only a few
compounds of the carbohydrate group, such as short and long chain β-fructans [FOS and inulin], lactulose,
and GOS, can be classified as prebiotics.
In 2008, the 6th Meeting of the International Scientific Association of Probiotics and Prebiotics (ISAPP)
defined “dietary prebiotics” as “a selectively fermented ingredient that results in specific changes in the
composition and/or activity of the gastrointestinal microbiota, thus conferring benefit(s) upon host health”.
There are also some revised definitions for prebiotics published in the scientific literature. However, the
above-mentioned definition, which was given in 2008, has been accepted in recent years. Despite the
absence of a consensus definition, the important part of the original and other definitions is that the
consumption of prebiotics is associated with human well-being. The word “selectivity”, or the potency of
a prebiotic to stimulate a specific gut microbiota, was another key element of the original definition;
however, this concept has been questioned recently.
In 2013, Scott et al. reported that the prebiotic effect was enhanced by cross-feeding, defined as the product
of one species which can be consumed by another one. This implication raises doubt for utilizing the
“selectivity” term in the prebiotics definition. A review on the evolution of prebiotics concept through
history can be found in a previous publication, and the debate on their definition is still ongoing.
Criteria:
The following criteria are used to classify a compound as a prebiotic:
 It should be resistant to acidic pH of stomach, cannot be hydrolyzed by mammalian enzymes, and
also should not be absorbed in the gastrointestinal tract.
 It can be fermented by intestinal microbiota.
 The growth and/or activity of the intestinal bacteria can be selectively stimulated by this compound
and this process improves host’s health.
Although not all the prebiotics are carbohydrates, the following two criteria can be exploited to distinguish
fiber from carbohydrate-derived prebiotics:
 Fibers are carbohydrates with a degree of polymerization (DP) equal or higher than 3 and
 Endogenous enzymes in the small intestine cannot hydrolyze them. It should be taken into account
that the fiber solubility or fermentability is not crucial
Types of Prebiotics:
There are many types of prebiotics. The majority of them are a subset of carbohydrate groups and are mostly
oligosaccharide carbohydrates (OSCs). The relevant articles are mainly on OSCs, but there are also some
pieces of evidence proving that prebiotics are not only carbohydrates.
• Fructans:
This category consists of inulin and fructo-oligosaccharide or oligofructose. Their structure is a
linear chain of fructose with β(2→1) linkage. They usually have terminal glucose units with
β(2→1) linkage. Inulin has DP of up to 60, while the DP of FOS is less than 10. Previously, some
studies implicated that fructans can stimulate lactic acid bacteria selectively. However, over recent
years, there are some investigations showing that the chain length of fructans is an important
criterion to determine which bacteria can ferment them. Therefore, other bacterial species can also
be promoted directly or indirectly by fructans.
• Galacto-Oligosaccharides:
Galacto-oligosaccharides (GOS), the product of lactose extension, are classified into two
subgroups: (i) the GOS with excess galactose at C3, C4 or C6 and (ii) the GOS manufactured from
lactose through enzymatic trans-glycosylation. The end product of this reaction is mainly a mixture
of tri- to pentasaccharides with galactose in β(1→6), β(1→3), and β(1→4) linkages. This type of
GOS is also termed as trans-galacto-oligosaccharides or TOS. GOSs can greatly stimulate
Bifidobacteria and Lactobacilli. Bifidobacteria in infants have shown high incorporation with GOS.
Enterobacteria, Bacteroidetes, and Firmicutes are also stimulated by GOS, but to a lesser extent
than Bifidobacteria. There are some GOSs derived from lactulose, the isomer of lactose. This
lactulose-derived GOSs are also considered as prebiotics [19]. Besides these types of GOS, the
other types are based on sucrose extension named raffinose family oligosaccharides (RFO). The
effect of RFO on gut microbiota has not been elucidated yet.
• Starch and Glucose-Derived Oligosaccharides:
There is a kind of starch that is resistant to the upper gut digestion known as resistant starch (RS).
RS can promote health by producing a high level of butyrate; so it has been suggested to be
classified as a prebiotic. Various groups of Firmicutes show the highest incorporation with a high
amount of RS. An in vitro study demonstrated that RS could also be degraded by Ruminococcus
bromii, and Bifidobacterium adolescentis, and also to a lesser extent by Eubacterium rectale and
Bacteroides thetaiotaomicron. However, in the mixed bacterial and fecal incubations, RS
degradation is impossible in the absence of R. bromii. Polydextrose is a glucose-derived
 oligosaccharide. It consists of glucan with a lot of branches and glycosidic linkages. There is some
evidence that it can stimulate Bifidobacteria, but it has not been confirmed yet.
• Other Oligosaccharides:
Some oligosaccharides are originated from a polysaccharide known as pectin. This type of
oligosaccharide is called pectic oligosaccharide (POS). They are based on the extension of
galacturonic acid (homogalacturonan) or rhamnose (rhamnogalacturonan I). The carboxyl groups
may be substituted with methyl esterification, and the structure can be acetylated at C2 or C3.
Various types of sugars (e.g., arabinose, galactose, and xylose) or ferulic acid are linked to the side
chains. Their structures vary significantly depending on the sources of POSs.
• Non-Carbohydrate Oligosaccharides:
Although carbohydrates are more likely to meet the criteria of prebiotics definition, there are some
compounds that are not classified as carbohydrates but are recommended to be classified as
prebiotics, such as cocoa-derived flavanols. In vivo and in vitro experiments demonstrate that
flavanols can stimulate lactic acid bacteria.
Thin Layer Chromatography (TLC):

TLC Protocol:
Theory: Thin layer chromatography is a method of analysis in which the stationary phase, a finely divided
solid, is spread as a thin layer on a rigid supporting plate; and the mobile phase, a liquid, is allowed to
migrate across the surface of the plate by means of capillary action.

Required material:
 Polymer (α glucan)
 TLC silica gel aluminum sheet
 Mobile phase
 Chromatogram
 Glucan and fructan spray (TLC stains)
 Distilled water
 Pipette
 Pencil and scale
 Dryer
 Cutter
 Measuring cylinder

Required chemicals:
Mobile phase: for exopolysaccarides
 Butanol 150ml
 Ethanol 150ml
 Water 90ml
Mobile phase recipe:
Mixed all the chemicals in a jar at the ratio of 5:5:3.

Procedure:
• Washed chromatogram properly then dried it.
• Measured the TLC silica gel aluminum sheet with the help of pencil and scale according to the
requirement.
• Cut the sheet with the cutter.
• Dissolved the polymer in distilled water and vortex it.
• Gently drawn a straight line across the plate approximately 1cm from the bottom (Did not use
excessive forces when wrote on a plate).
• Took 2µl from sample with pipette then spotted at the space of 1cm.
• After all spots had been applied, then dried the TLC sheet in dryer.
• Placed the sheet in the chromatogram and capped immediately to avoid loss of solvent.
• Waited for the 3 hour.
• Once the solvent reaches the top line on the sheet, removed it and allowed the sheet to dry.
Staining protocol:
Required material:
 Bottle
 Pot
 flame
 Distilled water
 Magnetic stirrer
 Measuring cylinder
 Beaker
 Aluminum foil
 Pipette
Required chemical:
 Water saturated butanol
 Analytical grade ethanol
 Phosphoric acid
 Urea
 Methanol
 Sulphuric acid

TLC stain fructan recipe:

Water saturated butanol preparation
Took 120ml butanol and 50ml water in a 500ml beaker. Placed it on magnetic stirrer for 15 to 20 minutes
covered the beaker with aluminum foil. After 20 minutes stopped the stirrer and let it rest for 5 minutes.
Used 5ml pipette to separate layer.
Took 100ml from water saturated butanol, 5ml analytical grade ethanol, 5.9ml phosphoric acid and 3g urea
and mixed them in a bottle.

TLC stain glucan recipe:

Took 95ml methanol and 5ml Sulphuric acid in a bottle.
Procedure:
• Poured the glucan stain in a pot.
• Placed the TLC sheet horizontally in the pot and immediately carried out.
• Dried with the help of dryer.
• Flamed until the solution shown the spot.
MRS Media:
De Man, Rogosa and Sharpe agar, often abbreviated to MRS, is a selective culture medium designed to
favour the luxuriant growth of Lactobacilli for lab study. Developed in 1960, this medium was named for
its inventors, Johannes Cornelis de Man, Morrison Rogosa, and Margaret Elisabeth Sharpe.

MRS Media Protocol:

Required material:
 Beaker
 Measuring cylinder
 Magnetic stirrer
 Pipette
 Autoclaved machine
 dis
 Test tubes
 Analytical balance
Required chemicals:

Composition Concentration g/L

Peptone 10g
Beef extract 8g
Yeast extract 4g
Dipotassium hydrogen phosphate (K2HPO4) 2g
Sodium acetate (CH3COONa) 3g
Diamoninum citrate 2g
Manganese sulphate (MnSO4) 0.05g
Magnesium sulphate (MgSO4) 0.2g
Sucrose 20%/glucose 2% 200g
Tween 80 1ml
Ph 6.2-6.4
Agar 15g

Procedure:
• Measured the amount of each chemicals on analytical balance.
• Mixed all required chemicals one by one in a beaker and put on the magnetic stirrer until they
dissolved.
• Maintained the pH 6.2-6.4.
• Raised the volume up to 1L with distilled water.
• Poured the media in the test tubes equal amount of 20ml.
• Then autoclaved.
Petri plate Pouring Protocol:
Required material:
 Laminar air flow
 Ethanol
 Glass burner lamp bottle
 Petri plates
 MRS media
 Wire loop
Procedure:
• Turned on the UV exposure for 15 minutes.
• Sterilized the laminar air flow with ethanol.
• Sterilized the hands with ethanol spray.
• Burned the flame.
• Lift the lid of a sterile petri plate.
• Carefully Poured liquefied MRS media into petri plates ensuring the entire bottom surface was
covered.
• Filled the plate to a depth that covered about one-third to one-half of its height.
• Then left the petri plates uncovered until it solidified under the laminar air flow.
• Once the agar had solidified and any moisture on the lid had evaporated, the plates sealed with their
lids.

Inoculation Protocol:
• Labelled the petri plates with relevant information.
• Sterilized the wire loop on flame until red hot.
• Obtained a small amount of the mixed microbial culture to be isolated.
• From the primary inoculum it was spread thinly over the plate by Streaking with the help of loop
in parallel lines (quadrant streaking).
• Incubated for 16 to 18 hours for DNA extraction and for 3 days for secondary metabolites.
Microscopy:
• Set the specimen appropriately by using adjuster and focus.
• Analyzed the slide at 100X with the help of immersion Oil.
Bacterial DNA Extraction
Required material:
 Pipette
 Centrifuge machine
 Incubator
 Proteinase k
 Vortex machine
 Water bath
 Eppendorf tube

Required Chemical and recipe:

 TE buffer
 CTAB/NaCl solution:
Dissolved 4.1g NaCl in 80ml distilled water. While stirring added 10g of CTAB to
dissolve, heat the solution at 65ċ and made the volume upto 100ml.
 10%SDS:
10g SDS was dissolved in 100ml distilled water.
 1M NaCl:
5.85g of NaCl dissolved in 100ml of distilled water.
 G+ve lysis buffer:
Tris HCL, EDTA, triton x 100,and freshly prepared lysozyme 20mg/ml.
 Phenol Chloroform Isoamyl (25:24:1)

Bacterial DNA Extraction Protocol:

• Made pellet of bacterial culture depending upon the growth (800µl, 1ml, 1.5ml) by centrifugation
at 12500rpm for 8 minutes.
• Discarded the supernatant and washed the pellet with 500µL of TE Buffer with repeated
pipetting to break pellet and centrifuge it again and discarded supernatant.
• Added 460µL of TE Buffer, repeated pipetting and added 100µL of G+ve Lysis Buffer, mixed it
and incubated at 37ċ for 30 minutes.
• Added 30µL SDS 10% and 8µL Proteinase K and vortex for 1 minute and incubated at 37ċ for 1
hour.
• In the meantime, put the CTAB/NaCl on water bath at 65ċ
• After incubation, added 100µL of 5M NaCl and 80µL CTAB/NaCl in it by repeated pipetting and
incubated it at 65ċ for 20 minutes.
• After incubation, added equal volume (780µL) of Chloroform:Isoamylalcohol (24:1) and mixed it,
then centrifuge for 56 minutes
• Separated the upper layer in a new eppendorf taking great care not to touch the interface (protip:
use yellow tips).
• Added 3-4µL of RNase A, mixed and incubated at 37ċ for 30 minutes.
• Added equal volume of Phenol: Chloroform: Isoamyl (25:24:1), mixed and centrifuge 6 minutes.
Separated the upper layer in new eppendorf.
• Added 0.6 volume of ice-cold isopropanol in it, mixed for sometime by gently inverting the tube
back and forth and then centrifuge it for 6 minutes. Discarded supernatant.
• Added 1mL of 70% ethanol in pellet and centrifuge it for 5-6 minutes. Discarded supernatant and
dried pellet. Did not over dried it
• Added 30ml autoclaved PCR H2O in it. Let it sat for 5-10 minutes, then ran on 1% Agarose gel.
 CONCLUSION
Our one month internship at the National institute of Biotechnology and Genetic Engineering, Faisalabad,
has been a truly explore with research projects and enlightening experience. Throughout this internship, we
had the chance to work with cutting-edge technologies and practices in analyzing vast amounts of genomic
data in health biotechnology, conducting experiments, and gaining invaluable insights into the field of two
different divisions: health biotechnology and industrial biotechnology division.
During our time at NIBGE, we actively participated in various specific experiment and research, making
significant contributions to the organization's ongoing initiatives. Practices capabilities allowed us to delve
into complex prebiotic and uncover new perspectives and discoveries. Its versatility proved to be a game-
changer, enabling us to process and interpret data efficiently, thus expediting the research process.
The hands-on experience we gained while working with state-of-the-art laboratory equipment and
techniques was incredibly valuable Participating in nanobiotechnology (synthesis of silver nano particles),
PCR, and such different experiment preparation provided us with a profound understanding of these
techniques and their applications in modern biotechnology and genetic engineering.
Beyond the technical skills we developed, the internship also honed our abilities in data analysis, problem-
solving, and critical thinking. We learned how to effectively communicate research findings and present
complex information to diverse audiences. This experience undoubtedly sharpened our professional
acumen and prepared us to tackle real-world challenges in the field of biotechnology and genetic
engineering.
Overall, our internship at NIBGE has been an incredible journey of growth and discovery. We are sincerely
grateful for the opportunity to contribute to the organization's scientific endeavors and expand our
knowledge in this field.
We extend our heartfelt gratitude to the entire team at NIBGE for their warm hospitality and unwavering
encouragement throughout our internship. The experience we gained during this period will undoubtedly
propel us towards making meaningful contributions to the advancement of science and technology in the
future.
Thank you