INTERNSHIP REPORT

Hands on training of Advanced Biotechnology

National Institute of Genomics and Advances Biotechnology
(NIGAB), National Agriculture Research Center Islamabad,
Pakistan
24 th June – 05th August 2024

SUBMITTED BY
Name: Eisha Zainab
(BIOT51F21S002)
Session 2021-2025
Department of Biotechnology,
Faculty of natural and applied sciences
University of Sargodha

Internship Supervisor: Dr.Kashif

Table of Contents
CERTIFICATION.........................................................................................................3
Acknowledgement.......................................................................................................4
Introduction to NARC,Islamabad.................................................................................5
Introduction to NIGAB.................................................................................................8
Laboratory Safety Rules............................................................................................11
Common Lab equipments at NIGAB.....................................................................13
EXPERIMENT 01 DNA EXTRACTION.....................................................................18
EXPERIMENT 2 GEL ELECTROPHORESIS...........................................................22
EXPERIMENT 03 RNA EXTRACTION.....................................................................24
EXPERIMENT 4 PCR...............................................................................................26
Addition of Master Mix.....................................................................................27
Tips and Considerations.................................................................................28
EXPERIMENT 5 DNA EXTRACTION BY KIT METHOD..........................................29
EXPERIMENT 06 CDNA SYNTHESIS.....................................................................31
EXPERIMENT 07 CLONING PCR............................................................................34
CERTIFICATION
This certifies that Isha Nasar successfully completed an internship in Molecular
Techniques at NIGAB, demonstrating exceptional proficiency in PCR, gel
electrophoresis, and DNA/RNA extraction. Her expertise and collaborative spirit
significantly enhanced our research projects. We commend her commitment and
extend our best wishes for her future endeavors.
Director
-------------------------
Dr.Sahu
kat Ali
Director
NIGAB
NARC,Is
lamabad

Supervisor
-------------------------
Dr. Sani
a Begum
Scientific
Officer,NIGAB
Acknowledgement

In the name of Allah, the Most Compassionate, the Most Merciful, I extend my
profound appreciation for the privilege of commencing my internship with NARC.

First and foremost, I extend my sincere appreciation to Director NIGAB, Dr. Shaukat
Ali, for his gracious approval, which granted me access to this esteemed research
institution.

I am profoundly grateful to Dr. Sania Begum for her exceptional mentorship and
unwavering support during my internship at the National Institute of Genomics and
Advanced Biotechnology (NIGAB). Her guidance not only deepened my
understanding of [specific field or project] but also significantly contributed to my
professional development. Dr. Sania’s expertise, patience, and encouragement were
invaluable throughout this transformative experience.

I also wish to convey my heartfelt thanks to Ma’am Zunaira and Sir Zeeshan Zafar
for their constant support and assistance during my time at NIGAB. Their dedication
to fostering a conducive learning environment greatly enhanced my overall
experience.

It has been an honor to work under the supervision of Dr. Sania Begum and
alongside Sir Zeeshan and Ma’am Zunaira. Their mentorship and support have been
instrumental in shaping my career aspirations, and I am truly grateful for their
guidance.

Introduction to NARC,Islamabad
The National Agricultural Research Centre (NARC) in Islamabad, Pakistan,
established in 1984, is a pivotal institution in the nation's agricultural research
landscape. Operating under the Pakistan Agricultural Research Council (PARC),
NARC is the premier entity for advancing agricultural productivity, sustainability, and
food security through cutting-edge research and technological innovation.
NARC serves as a collaborative hub, bringing together scientists, researchers, and
agricultural specialists to tackle Pakistan's agricultural challenges. Its research spans
crop sciences, livestock management, horticulture, natural resource management,
biotechnology, and agro-processing. With a network of research institutes,
experimental stations, and laboratories nationwide, NARC effectively disseminates
its findings and technologies to farmers and stakeholders, significantly contributing to
agricultural progress and rural development.

NARC's commitment to research excellence is reflected in its numerous accolades,

strategic partnerships, and influential role in shaping agricultural policies and
providing advisory services. As Pakistan faces emerging agricultural challenges,
NARC remains at the forefront, dedicated to leveraging science and technology for
sustainable agricultural growth and national food security.

Additionally, NARC boasts extensive infrastructure, including 58 specialized

research laboratories, a National Gene Bank, a comprehensive agricultural sciences
library, a national herbarium, a grain quality testing laboratory, an auditorium,
hostels, workshops, a cafeteria, and audiovisual studios, all designed to foster
innovation and collaboration in agricultural research.
Research Objectives
Enhancing Productivity
This study endeavors to pioneer cutting-edge biotechnological strategies
aimed at revolutionizing agricultural productivity. By leveraging advancements in
genetic engineering, molecular breeding, and bio-stimulants, the research aspires to
significantly elevate crop yields, strengthen resilience to environmental challenges,
and maximize resource efficiency.

Through meticulous experimentation, this research seeks to uncover transformative

insights and actionable solutions for sustainable agriculture, with the ultimate goal of
advancing global food security and empowering farmers to cultivate a more resilient
and prosperous future.

Innovative Techniques
By integrating cutting-edge technologies such as nanotechnology and
precision agriculture, these innovative techniques are poised to transform traditional
practices fundamentally. These advancements are designed to maximize resource
efficiency, enhance productivity, and promote sustainability across various
industries. With ongoing research and development, these approaches hold the
potential to offer sophisticated solutions to the complex challenges facing global
agriculture today.

Agricultural Advancements
Agricultural advancements aim to secure global food supply, foster
sustainability, and enhance economic viability through innovative technologies and
practices. By leveraging precision farming, biotechnology, and digital agriculture,
these innovations seek to maximize crop yields, reduce environmental impact, and
elevate farmers' livelihoods worldwide. Ongoing research and application of these
advancements are critical in tackling global challenges, ensuring a resilient and
sustainable food production system for future generations.

Sustainable Resource Management and Ecological Preservations

Sustainable resource management focuses on efficiently using natural
resources while preserving the environment and meeting societal needs. By
integrating conservation practices, renewable energy, and ecosystem management,
it aims to balance economic growth with environmental protection, ensuring
resilience and equitable access for current and future generations.
Introduction to NIGAB
The National Institute for Genomics and Advanced Biotechnology (NIGAB) is
a leading research institution in Pakistan, dedicated to pioneering advancements in
biotechnology and genomics. Founded by Dr. Ghulam Ali, NIGAB is at the forefront
of genetic and biotechnological research in the region. Its mission is to drive
scientific innovation, promote educational excellence, and facilitate interdisciplinary
collaboration. Spanning 3,500 square meters, NIGAB boasts 28 laboratories, an
animal house, and Biosecurity Level 3 facilities. This state-of-the-art infrastructure
supports high-tech research aimed at ensuring national food security through
advanced biotechnological solutions.

Infrastructure
NIGAB boasts state-of-the-art infrastructure equipped with advanced
laboratories, research facilities, and specialized equipment. The institute's
infrastructure supports a wide range of research activities spanning genomics,
molecular biology, bioinformatics, and biotechnology applications.

Research Focus areas

At its core, NIGAB is devoted to a broad spectrum of research areas within
genetics and biotechnology, including:

 Enhance Crop Genetics:

Studying plant genomes to improve crop yield, resilience to diseases, and
adaptation to environmental stressors.
 Biomedical Applications:
Investigating genetic factors contributing to diseases prevalent in the region and
developing diagnostic tools and therapeutic solutions.
 Biodiversity Conservation:
Analyzing genomic diversity in native species to inform conservation
strategies and sustainable use of biodiversity.
 Bioinformatics:
Developing computational tools and algorithms for analyzing large-scale
genomic data, enhancing biological discoveries and applications.
Research Initiatives and Projects:

 Crop Genomics:
NIGAB undertakes research on key crops in Pakistan, including wheat,
rice, cotton, and maize, to pinpoint advantageous traits and enhance breeding
programs through genomic selection and genetic engineering.
 Genomics:
Research encompasses the investigation of genetic markers linked to
widespread diseases such as diabetes, cardiovascular disorders, and specific
cancers prevalent in Pakistani populations.
 Microbial Genomics:

Investigating the genetic diversity of microorganisms for potential applications

in agriculture, industry, and environmental remediation.
Technological Infrastructure:

NIGAB is equipped with state-of-the-art genomic technologies including:

 Next-Generation Sequencing (NGS) Platforms: For high-throughput

sequencing of genomes, transcriptomes, and epigenomes.

 Genome Editing Tools: Such as CRISPR-Cas9 for precise genetic

modifications in crops and model organisms.

 Bioinformatics Resources: Computational infrastructure for data storage,

analysis, and interpretation of genomic data.

Collaborations and Partnerships:

NIGAB engages in dynamic collaborations with top national and international

research institutions, universities, and industry leaders to advance genomic
research, facilitate knowledge transfer, and drive pioneering technological
innovations.

Impact and Future Directions:

The genomic research at NIGAB contributes to:

 Improved Agricultural Productivity: Through development of disease-

resistant and high-yielding crop varieties.

 Precision Medicine: Tailoring healthcare interventions based on genetic

profiles to improve disease prevention, diagnosis, and treatment outcomes.

 Conservation and Sustainability: Preserving genetic diversity and

promoting sustainable use of natural resources.

Facilities at NIGAB:

I. DNA and RNA Extraction

II. DNA Analysis, Gel Electrophoresis, Gel Documentation

III. PCR, RT-PCR, DNA Sequencing

IV. Ultracentrifugation, HPLC

V. V. Biosafety Level 3 Cabinet

VI. Qualitative and Quantitative ELISA

VII. GMO Detection, Tissue Culture

VIII. Transformation Protocols via Agrobacterium tumefaciens and Gene Gun

IX. Protein Analysis, 2-D SDS-PAGE

X. X. Greenhouse for Controlled Growth

XI. Aromatic and Non-Aromatic Rice Detection

Achievements of NIGAB

i. Transgenic Plants
ii. Rust resistant transgenic wheat line
iii. Drought and salt tolerant wheat lines
iv. Drought and salt tolerant rice lines
v. Herbicide resistant Groundnut transgenic lines
vi. Cold tolerant transgenic tomatoes
vii. Drought and salt tolerant maize lines

LABS at NIGAB

PLANT BIOTECHNOLOGY

i. Plant Genomics lab

ii. Genome editing
iii. Gene cloning
iv. Functional Genomics
v. Growth room
vi. Inoculation Lab
vii. Gene transformation Lab
viii. Gel electrophoresis Room
ix. Green super rice GSR

ANIMAL BIOTECHNOLOGY

i. ELISA testing lab

ii. Microbial Biotechnology
 iii. Animal Probiotics Lab
iv. Cell culture Lab
v. Animal Genomics Lab
vi. Health Biotechnology Lab
vii. Animal Containment
viii. Recombinant DNA Technology Lab

Throughout my six-week internship, I engaged in advanced projects within the Gene

Cloning, Genome Editing, and Functional Genomics laboratories. During this period,
I acquired comprehensive knowledge and hands-on experience with various
methodologies, tools, and techniques pertinent to plant biotechnology.
Laboratory Safety Rules
Laboratory safety is crucial for safeguarding personnel, protecting the
environment, and upholding the integrity of scientific research. Strict adherence to
safety protocols minimizes risks from hazardous chemicals, equipment, and
procedures. By fostering a culture of responsibility and awareness, effective safety
measures ensure a secure working environment that supports scientific excellence.

 Personal Protective Equipment (PPE):

 Wear appropriate PPE including lab coats, safety goggles, gloves, and
closed-toe shoes at all times in the laboratory.

 Ensure PPE fits properly and is in good condition to provide adequate

protection.

 Chemical Handling and Storage:

 Understand the properties and hazards of chemicals before use.

 Store chemicals following compatibility and segregation guidelines, ensuring

proper labeling and secure containment.

 Equipment Operation:

 Receive training on the operation of laboratory equipment and adhere to

manufacturer’s instructions.

 Conduct regular inspections of equipment for safety hazards and malfunction;

report any issues promptly.

 Emergency Procedures:

 Know the location and proper use of emergency equipment such as fire
extinguishers, eyewash stations, and safety showers.

 Understand emergency evacuation routes and procedures for spills, fires, and
other incidents.

 Laboratory Hygiene:

 Sustain a clean and orderly workspace to reduce clutter and mitigate potential
hazards.
 Dispose of chemical waste and hazardous materials in strict adherence to
established protocols and regulatory standards.
Personal Conduct:

 Avoid eating, drinking, or applying cosmetics in the laboratory to prevent

ingestion of hazardous substances.

 Refrain from performing unauthorized experiments or altering procedures

without apropriate authorization and risk assessment.

 Risk Assessment and Communication:

 Conduct thorough risk assessments before starting experiments and

implement appropriate safety controls.

 Communicate potential hazards, safety protocols, and emergency procedures

to all personnel involved in laboratory activities.

 Training and Education:

 Participate in laboratory safety training sessions and ongoing education

programs.

 Stay informed about updates in safety regulations, best practices, and

technological advancements relevant to laboratory operations.

 Incident Reporting and Investigation:

 Report all accidents, near misses, and safety concerns to laboratory

management or designated safety officers.

 Cooperate in incident investigations to identify root causes and implement

corrective actions to prevent recurrence.

 Continuous Improvement:

 Consistently review and update safety protocols based on lessons learned,

new research findings, and regulatory changes.

 Cultivate a culture of safety awareness and accountability among all

laboratory personnel through open communication and collaboration.

 Ventilation and Air Quality:

 Make sure there is enough ventilation in the laboratory to reduce exposure to

airborne contaminants and keep air quality safe.
  Use fume hoods when working with volatile chemicals, toxic substances, or
any materials that can produce harmful fumes or vapors.

 Electrical Safety:

 Inspect electrical cords, plugs, and equipment regularly for damage or wear;
replace faulty equipment immediately.

 Avoid overloading electrical outlets and use ground fault circuit interrupters
(GFCIs) where required.

 Biological Safety:

 Follow biosafety rules when handling biological materials, like microorganisms,

genetically modified organisms (GMOs), and human or animal tissues.

 Use the right safety measures (such as biosafety cabinets and personal
protective equipment) based on the risks associated with the biological agents.

 Radiation Safety:

 Follow radiation safety guidelines for handling radioactive materials and use
appropriate shielding, monitoring devices, and personal protective equipment.

 Limit exposure time

Common Lab Equipments at NIGAB

PCR MACHINE
PCR stands for polymerase chain reaction. A PCR machine is used to make
many copies of a specific segment of DNA, ranging from thousands to millions. There
is also an RT-PCR machine available, which is used for reverse transcription PCR,
allowing the amplification of RNA.

Laminar flow cabinet

A laminar flow cabinet is a special workspace used in labs to create a clean
and sterile environment for handling sensitive materials. It uses HEPA filters to
remove airborne contaminants and provides a steady flow of clean air over the
working area. These cabinets are important for tasks that need to be free from
contamination, such as cell culture and microbiological work, helping to keep
experiments and samples safe.
Centrifuge Machine
Centrifuge machines are vital laboratory instruments that expertly separate
substances of varying densities and sizes by spinning them at high speeds. These
powerful devices play a crucial role in numerous applications, including the
separation of blood components, DNA extraction, and the isolation of cellular
components in both biological and chemical research. Their ability to efficiently and
effectively isolate materials makes them indispensable tools in modern scientific
exploration.

Magnetic stirrer

A magnetic stirrer is an advanced laboratory apparatus that creates a rotating

magnetic field to stir solutions through a magnetic stir bar suspended within the
container. Renowned for its versatility, this device is indispensable in the mixing of
liquids, the dissolution of solids, and the preservation of uniformity in both chemical
reactions and biological assays. Its precise control and operational efficiency render
it a crucial instrument in a myriad of scientific endeavors.

Gel Documentation System

A Gel Documentation System is a specialized imaging device used in

molecular biology labs to capture and analyze images of nucleic acid and protein
gels. It features UV light for visualizing gels, digital imaging to take high-resolution
pictures, and software for analyzing and documenting gel electrophoresis results.
These systems are essential for studying DNA, RNA, and protein samples in
research, diagnostics, and forensic applications.

PH meter
A pH meter is a scientific instrument used to measure the acidity or alkalinity of a
liquid. It provides accurate pH readings based on the concentration of hydrogen ions
present in the solution.

Water Bath
 A water bath is a laboratory device used to heat samples or maintain a
constant temperature in a controlled environment. It consists of a container filled with
water that is heated or cooled as needed, providing gentle and uniform heat transfer
for incubation, melting, and general heating applications in scientific experiments and
medical procedures.

Weighing Balance
A weighing balance is a precise instrument used to measure the mass of
objects or substances with high accuracy and sensitivity, essential in scientific
research, pharmaceuticals, and quality control processes.

Pipette

A pipette is a laboratory tool used to transfer precise volumes of liquids,

crucial for accurate measurement in scientific experiments and medical procedures.
Ultra pure water machine

An ultra-pure water system represents a state-of-the-art purification technology

that integrates sophisticated methods such as reverse osmosis, deionization, and
advanced filtration to produce water of unparalleled purity, free from contaminants
and minerals. This meticulously refined water is essential for laboratories engaged in
delicate tasks, including analytical chemistry, molecular biology, and semiconductor
fabrication. By upholding the highest purity standards, these systems enable
researchers and manufacturers to obtain precise outcomes and preserve the
integrity of their work.

Autoclave

An autoclave is a sophisticated sterilization apparatus that utilizes high-

pressure steam to eliminate microorganisms and pathogens from equipment,
glassware, and biological waste. By reaching temperatures well beyond the boiling
point of water, it ensures comprehensive sterilization, rendering it indispensable in
laboratories, medical facilities, and industrial settings. Autoclaves are crucial for
maintaining sterility and preventing contamination, thereby playing a pivotal role in
adhering to stringent sterilization standards and ensuring safety. Their dependability
and efficacy are essential in any environment where hygiene and precision are of
utmost importance.
Microwave oven

In a laboratory setting, a microwave oven is sometimes used for specific

scientific purposes such as:

 Heating or thawing biological samples or reagents quickly and uniformly.

 Assisting in certain chemical reactions that require controlled heating.

 Sterilizing laboratory glassware and equipment, especially when autoclaving

is not feasible.

However, it's important to note that microwave ovens used in labs are often
designated for specific scientific applications and are not the same as standard
kitchen microwave ovens.

Vortex Mixer
An autoclave is a powerful sterilization device that employs high-pressure
steam to eradicate microorganisms and pathogens from equipment, glassware, and
biological waste. By achieving temperatures significantly above boiling point, it
guarantees thorough sterilization, making it indispensable in laboratories, medical
facilities, and industrial environments. Autoclaves are essential for upholding sterility
and preventing contamination, and they play a critical role in ensuring safety and
compliance with rigorous sterilization standards. Their reliability and effectiveness
are non-negotiable for any setting where hygiene and precision are paramount.

EXPERIMENT 01 DNA EXTRACTION

APPARATUS

 Eppendorf tube
 Water bath
 Leaf sample
 Pestle mortar
 Pipette
 Centrifuge machine
 Vortex

Chemicals Required

 DNA extraction buffer

 70 %Ethanol
 KAC buffer
 Isopropanol

Protocols of Chemicals

i. DNA stock solution

0.5M EDTA _10 ML
Tris Base_6.05g
KCL_37.85g
PH _9.5
Double Distilled water _400ML
 TOTAL VOLUME 500ML
ii. DNA extraction buffer
Stock DNA buffer _50ML
PVP _0.02g
Na2 So3 _0.180g
iii. 0.5M EDTA
Na2 EDTA 2 H20 _93.05g
Double distilled water _400ML
PH _8
PROCEDURE

1. Sample Collection

- Conduct under sterile conditions.

- For wheat super-breeding, meticulously excise leaves from wheat plantlets grown
under controlled laboratory conditions.

- Place each leaf sample into a separate Eppendorf tube and label accordingly.

2. Pre-Heating

- Preheat the extraction buffer to 65°C for 10-12 minutes using a water bath.

3. Equipment Cleaning

- Disinfect pestles, mortars, and other tools by spraying with 70% ethanol, followed
by wiping and drying with tissue.

- Handle the leaf samples with care, spray with ethanol, and sterilize both the leaf
and gloved hands.

4. Leaf Grinding

- In a mortar, add 1 ml of DNA buffer and finely grind the leaf tissue until a green
liquid is formed.

5. Sample Transfer

- Transfer the sample from the mortar to a 1.5 ml Eppendorf tube using a pipette.

- If the volume is less than 1 ml, adjust to 1 ml by adding additional DNA buffer.

6. Sample Heating

- Incubate the sample at 65°C for 40-60 minutes.

7. Vortexing

- Vortex each sample for 5 seconds every 10 minutes.

8. Addition of KAC Buffer

- Allow the sample to reach room temperature, then add 0.3 M KAC buffer.

- Incubate for 30 minutes.

9. Centrifugation 1

- Centrifuge at 12,000 rpm for 10 minutes.

10. Sample Replication

- Divide each sample into two 500 µl aliquots.

11. Addition of Isopropanol

- Add 420 µl of chilled isopropanol to each sample.

12. Incubation

- Incubate at -20°C for 1 hour.

13. Centrifugation 2

- Centrifuge at 12,000 rpm for 10 minutes.

- Carefully decant the supernatant and retain the pellet.

14. Pellet Washing

- Wash the pellet with 50 µl of 70% ethanol.

15. Centrifugation 3

- Centrifuge at 12,000 rpm for 5 minutes.

- Retain the pellet.

16. Final Pellet Washing

- Wash the pellet again with 70% ethanol and discard the supernatant.

17. Centrifugation 4

- Centrifuge at 12,000 rpm for 5 minutes.

18. Sample Drying

- Invert the tube to dry the pellet on absorbent paper.

- Dissolve the pellet in 20-50 µl of double-distilled water, depending on the

concentration of the sample.

- The samples are now prepared for subsequent processing through gel
electrophoresis.
EXPERIMENT 2 GEL ELECTROPHORESIS
Apparatus

 50X TAE
 Double distilled water
 Agarose
 Ethidium Bromide
 Paraffin sheet
  Oven
 Loading Dye
 Electrophoresis tray and tank

Protocols of Chemicals

I. 1X TAE
Double distilled water _980ML
50X TAE_20ML
Total volume _1000 ML-1liter
II. 1%Agarose Gel
 1X TAE 40ML
 AGAROSE 0.4g
 Ethidium Bromide 4 microliter
III. 2% Agarose Gel
 1X TAE 40ML
 Agarose 40ML
 Ethidium Bromide 7 microliter
PROCEDURE

1. Take 40ML 1X TAE in flask.

2. Add 0.4g agarose measured on balance.
3. Pre heat in oven for 1.5 minute in oven, while preparing tray.
4. Cool at room temperature.
5. Add 4 microliter Ethidium Bromide. Mix well and pour immediately pour in
tray.
6. Place at room temperature for 10 – 15 minutes to let gel solidify.
7. Remove stoppers and combs.
8. Place in tank containing TAE buffer.
9. LOADING DYE, Using pipette add 2 microliter on paraffin sheet in forms of
dots.
10. Loading dye used should be half of the sample conc.
11. Pick sample 4 microliter ,mix with loading dye using pipette and place in
solidified gel wells.
12. Apply electric field as following
 100 voltage
 300 current
 20-30 minutes
Now place gel in GEL DOC to view bands.
EXPERIMENT 03 RNA EXTRACTION
APPARTUS

 Liquid Nitrogen
 Trizol Reagent
 Chloroform
 70%Ethanol
 Al Foil

PROCEDURE

1. Sample Collection
 Sterilize gloved hands ,scissors and leaf node.
 Growing leaf is preferred for RNA extraction.
 Wrap leaf in sterilized Aluminium foil and label it with sample no.
 Wrap immediately and place in liquid nitrogen.
2. Crushing of sample
 Take and sterilize mortar pestle with 70 %ethanol .
 Now take sample from liquid nitrogen and crush it well.
 During crushing liq N2 should be poured continuously in order to crush
sample until converted to fine powder.
3. Addition of chilled trizol
 Take 1ml chilled TRIZOL and add to Eppendorf tube.
 Mix it well incubate in ice for 10 minutes.
4. Centrifuge1 at -4c for 10 minutes at 12,000rpm.
 After centrifugation place in ice.
 Pick supernatant 700microliter in separate labelled tube.
 Place in ice afterwards.
5. Addition of chilled choloroform
 Take 300microliter and add.
 Incubation in ice for 10 minutes.
6. Centrifuge 2 for 15 minutes at -4c,12,000rpm.
 Take palette and discard supernatant 300 microliter.
7. Addition of chilled isopropanol
 Add 420microliter.
 Invert ependroff to mix well.
 Incubate in ice for 10 minutes.
8. Centrifugation 3 for 10minutes at -4c for 12,000rpm.
 Discard supernatant ,place in ice.
9. Washing with ethanol
 Take 1ML 70%ethanol and wash palette
 Mix it well.
10. Centrifuge 4 for 5minutes at -4c 12,000rpm.
 Place in ice.
 Wash again with 70%ethanol.
 Discard supernatant and take palette layer.
11. Centrifuge 5 for 5 minutes at -4C,12,000rpm.
12. Air drying for 1 minute.
 Remove ethanol by pipette.

13.Dilution with Double distilled water add 10microliter to dissolve palette.

14.Gel Electrophoresis run for visualization of bands.

15.Incubation at -80c for storage.

EXPERIMENT 4 PCR
Preparation of master mix.

 Master mix should 18microliter and DNA sample should be 2microliter.

 Total volume should be 20 microliters, for cross checking add all values and
divide buy total no of samples.
 DNA used for PCR should be of same variety.
 DNA quantity should remain 2 microliters despite the no of samples.
 1-2microliters extra master mix should be prepared.

Chemicals Quantities 10Xsamples

H2O 9 99
RT buffer 5 55
MgCl2 2 22
NTPs 0.5 5
Forward primer 0.5 5
Reverse primer 0.5 5
Taq polymerase 0.5 5

 Why these chemicals are added.

 A. RT BUFFER. For efficient Cdna synthesis.
 To prevent degradation.
B. MgCl2. Activates DNA polymerase.
 DNTP binding.
 Stabilization of DNA polymerase.
C. NTPs. To expand the growing strands.
 The triphosphate part of each NTP molecule (three phosphate groups)
provides the energy needed for the polymerization reaction.
D. Forward primer.
 Target DNA Recognition
 Initiation of DNA Synthesis
E. Reverse primer.
 Defining Amplification Region
 The reverse primer ensures that DNA synthesis occurs in the correct direction
relative to the target DNA sequence.
F. Taq polymerase.
 Amplification of Long DNA Fragments
 Taq polymerase uses deoxyribonucleotide triphosphates (dNTPs) as
substrates to extend the primers and synthesize new DNA strands.

Cycles of PCR

1) 94C-------5minutes
2) 95C-------45sec
3) 58c--------90sec
4) 72---------60sec

Dilution of primers

 20-80 concentration
 E.g if primer 20microliters primer add 80microlioters double distilled water.
  Take 20microliters primer inn Eppendorf tube ,place in ice, add 80microliters
water.

 Calculate Primer Concentration: Determine the concentration of your

primer stock solution. Primers are typically synthesized and provided in a
concentrated form (e.g., 10 µM).

 Calculate Dilution Factor: Decide on the final concentration of primers in

your PCR reaction mix. This can vary depending on the specific protocol and
optimization requirements. A common starting concentration is around 0.1-1
µM for each primer in the reaction.

 Prepare Dilution: Use molecular biology-grade water (nuclease-free water)

to dilute the primer stock solution to the desired concentration. For example,
if you have a 10 µM primer stock and want a final concentration of 1 µM, you
would dilute it 1:10 (1 part primer stock + 9 parts water).

 Mix Thoroughly: After dilution, vortex or gently mix the primer solution to
ensure homogeneity.

Addition of Master Mix

1. Prepare Master Mix: The master mix typically contains all components
needed for PCR, except the template DNA and primers. This includes PCR
buffer, dNTPs (nucleotides), and DNA polymerase (such as Taq polymerase).

2. Calculate Volume: Calculate the total volume of master mix needed per
reaction. This will depend on the number of reactions you are setting up and
any pipetting margin for error.

3. Mix Components: Thoroughly mix the master mix components by gently

vertexing or pipetting up and down. It’s important to ensure all components
are evenly distributed throughout the mix.

4. Add Primers: After preparing the master mix, add the appropriate volume of
each diluted primer solution to achieve the final desired concentration in each
PCR reaction. For example, if your protocol calls for a final concentration of
0.5 µM for each primer and you are setting up a 25 µL reaction, you would
add 1 µL of the diluted primer solution (0.5 µM concentration) to each reaction
tube.
 5. Mix Again: Gently mix the reaction tubes after adding the primers to ensure
uniform distribution of all components.

Tips and Considerations

 Avoid Contamination: Use sterile techniques and nuclease-free reagents
and tubes to prevent contamination of your PCR reactions, which can lead to
erroneous results.

 Pipetting: Use accurate pipettes and calibrate them regularly to ensure

precise measurement of small volumes, especially when working with low
concentrations like primers.

 Temperature Control: Keep all reagents, especially enzymes like Taq

polymerase, on ice or at the recommended storage temperature until ready to
use to maintain their stability and activity.

PCR PROCESS

 Denaturation: The first step of PCR involves heating the reaction mixture to a
high temperature (typically around 94-98°C). This heat causes the double-
stranded DNA template to separate (denature) into two single strands.

 Annealing: After denaturation, the reaction temperature is lowered to allow

the primers to anneal (bind) to their complementary sequences on the single-
stranded DNA template. The annealing temperature typically ranges from 50-
65°C, depending on the melting temperature (Tm) of the primers.

 Extension (Elongation): Once the primers are bound, the reaction

temperature is increased again, usually to around 72°C. At this temperature,
DNA polymerase, often Taq polymerase, extends each primer by adding
complementary nucleotides to the template strand. Taq polymerase
synthesizes new DNA strands in the 5' to 3' direction, using the single-
stranded DNA as a template.
  Repeat Cycles: Steps 1 to 3 (denaturation, annealing, and extension)
constitute one PCR cycle. Typically, PCR is performed through multiple cycles
(20-40 cycles or more), each lasting around 1-3 minutes depending on the
length of the DNA fragment being amplified and the efficiency of the
polymerase used.

 Final Extension: After the last cycle, a final extension step is often included
where the reaction is held at 72°C for an additional 5-10 minutes. This allows
any remaining DNA strands to complete their synthesis

EXPERIMENT 5 DNA EXTRACTION BY KIT METHOD

Kit Components

 LYSIS BUFFER break open the cells to release DNA.

 PROTEINASE K degrade proteins.
 BINDING BUFFER helps DNA to bind to silica.
 WASH BUFFER removes impurities and containments from DNA.
 ELUTION BUFFER release DNA.
 ISOPROPNAOL/ETHANOL precipitates DNA from solution.
 COLLECTION TUBE /SPIN COLUMN holds samples during extraction.
 CENTRIFUGE TUBE used for washing and centrifugation of DNA
 RNAase A,B removes DNA from sample.
 LOADING DYE facilitates DNA loading onto gel or PCR.

PROCEDURE

1. Sample collection
 Weigh plant tissue -use upto 100mg of fresh tissue or 20mg of frozen tissues.
2. Grinding of sample
a) Mortar and pestle place plant tissue and liquid N2 and grind it thoroughly.
b) Grinding Mill place up to 100mg of tissues in a vial containing stainless
steel beads. The vial and beads should be pre-cooled with liq N2.
3. Picking up sample
 Transfer powdered tissues with 1.5ml microcentrifuge tube containing
350microliters of lysis buffer A.
4. Vortex for 10-20sec.
5. Lysis buffer A
 Add 50 microliters and 20 microliters RNAse A.
6.Incubation
 of sample at 65c for 10 minutes, vortex occasionally.
7.Precipitation solution
 Add 130microlitersand mix by inverting the tube for 2-3 minutes. Incubate in
ice for 5 minutes.
8.Centrifuge 1 for 5 minutes at >20,000rpm—g (>14,000rpm).
9.Collection of supernatant 400microliters and transfer to a clean
microcentrifuge tube.
10.Addition of binding solution 400 microliters and 400 microliters ethanol.
11.Replicate of Mixture
 Transfer half of prepared mixture 600 microliters in spin column.
12.Centrifuge 2
 For 1 minute at 6000 –g(8000rpm)
 Discard flow through solution and apply remaining mixture onto the same
column.
13.Centrifuge 3
 For 1 minute at 6000-8000rpm.
14.Addition of wash buffer 1 to column 500 microliters.
15.Centrifufge 4 for 1 minute .
 Discard flow through and place the column back to centrifuge tube.
16.Addition of wash buffer 2, 500 microliters to column
17.Centrifuge 5 for 3 minute at max >20,000-->14,000 rpm.
 Discard collection tube containing flow through and transfer the column to
sterile 1.5ml microcentrifuge tube.
 18.Addition of elution buffer 100 microliters to centre of column
membrane .
 Incubate for 5 minutes.
19.Centrifuge for 1 minute.
20.Again, elution can be performed.
 Store at -20C.
EXPERIMENT 06 CDNA SYNTHESIS

 Synthetic DNA molecule, complementary to a specific RNA molecule e.g

mRNA.
 Created by reverse transcription and RNA conc.is checked by Nanodrop.
 RNA conce should be 20microliters.

RNA conc. 2d H2O Oligo DNTP Master mix

2.5 9.5 1 7

10.75 10.75 1 7

10.68 10.68 1 7

Master Mix RNA +DH2O+Oligo

DNTP^d 2microliters

RT buffer 4microliters

RT enzyme 4microliters

Outline:

1. RNA Extraction
 Isolate total RNA from the tissue or cell samples.
 Use appropriate kits or reagents for RNA extraction (e.g., TRIzol reagent).
 Ensure RNA quality and purity using spectrophotometry (e.g., A260/A280
ratio).
2. RNA Quality Check
 Run extracted RNA on an agarose gel to check integrity.
 Use a bioanalyzer for precise assessment of RNA integrity number (RIN).
3. DNase Treatment
 Treat the RNA samples with DNase to remove any contaminating genomic
DNA.
 Purify the RNA post-DNase treatment.
4. Reverse Transcription
 Set up the reverse transcription (RT) reaction using a reverse transcriptase
enzyme.
 Convert purified RNA into cdna using enzyme.
 Primers are annealed to to RNA template.
5. Second strand synthesis
 Of DNA using DNA polymerase enzyme.
6. cDNA Quality Check
 Dilute the cDNA sample as required.
 Check cDNA quality and concentration using spectrophotometry or a
fluorometer.
 Optionally, perform a PCR with housekeeping gene primers to verify
successful reverse transcription.
7. CDNA Analysis
 Analyze PCR or qPCR results to determine the relative expression levels of
target genes.
 Normalize gene expression using housekeeping genes as controls.
 Use appropriate software tools for data analysis (e.g., ΔΔCt method for
qPCR).
8. Documentation and Reporting
 Document all experimental conditions, reagent details, and protocols used.
 Report the results with appropriate controls, replicates, and statistical
analysis.

Procedure

1. Take PCR voiles and add RNA ,2DH2O and oligo DNTP.
2. Apply PCR for 5 minutes at 65C ,incubate in ice.
3. Apply PCR for 1.5hr at 42C.
4. Again PCR for 10minutes at 72C.
5. The cDNA is synthesized.

RT-PCR
  Add control (actin),primer (0.5)forward and reverse and master mix (5)and
cdna (1)
 All quantities in in microliters.
 Make 92 well plate and apply RT PCR and analyze the results.

EXPERIMENT 07 CLONING PCR

Apparatus

 T.E buffer(Tris +EDTA)

 Bacterial colony
 Ice to incubate primer
 Prepared master mix
 Forward and reverse primer
 Lysozyme buffer
 PCR water
DNA extraction methods

I. Physical methods
II. Chemical methods
III. Enzymatic methods
IV. Kit methods

Procedure

1. Take 35 microliters T.E buffer in Eppendorf tube.

 Take bacterial colony and add in buffer.
 Make it turbid by toothpick to dissolve.
 Label the tube.
2. Denaturation
 External temperature 95C in PCR machine.
3. Centrifugation
 At 6000 rpm for 6 minutes.
4. Add master mix 25 microliters.
5. ADD PCR water 12 microliters.
6. Template DNA (supernatant of PCR sample)
7. 2.5 microliters forward and reverse primer respectively.
8. Vortex for 5 sec
9. Complete PCR cycles.
10. Prepare 2% agarose gel ,load sample .
11. Run gel electrophoresis and view bands on gel doc.