Internship Report 2023

Agricultural Biotechnology Research Institute, AARI Faisalabad

Submitted by: Sumraiz Ashraf

(2019-ag-6379)

A dissertation submitted in partial fulfillment of the requirements for the

Degree: B.Sc. (Hons.) Agriculture Sciences

Department : Plant Breeding and Genetics

UNIVERSITY OF AGRICULTURE, FAISALABAD, Sub Campus Burewala

1
 DEDICATION

Dedicated to
MY BELOVED PARENTS
Who taught me
the first word to speak,
the first alphabet to write
and the first step to take&
&

MY HONOURABLE TEACHERS
Who guided me at each and every step during my studies

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 Certification

It is certified that Sumraiz Ashraf S/O Muhammad Ashraf, Reg. # 2019-ag-6379 has worked
in Agricultural Biotechnology Research Institute (ABRI) Faisalabad from 15-02-23 to 28-04-23
as a part of internship program.He has performed his work successfully and his report has
processed for evaluation.

Chief Scientist ABRI, Faisalabad _____________________

Dr. Sajid Ur Rehman

Internship station Supervisor ______________________

(Senior Scientist ABRI, Faisalabad ) M. Waqas Jamil

Internal Supervisor _____________________

(Internship Incharge) Dr. Muhammad Shaban
(PBG Department)
UAF sub-campus, Burewala

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 ACKNOWLEDGEMENT

Numerous thanks to Dr. Muhammad Shaban (Internship In-charge) for his kind action to
manage my Internship at Agricultural Biotechnology Research Institute (ABRI) Faisalabad. And
thanks to Dr. M. AbubakkarAzmat, head of department Plant Breeding & Genetics for his
kindness and guidance for the selection of Institute for completion of my Internship.

The work presented in this manuscript was accomplished under the enlightened supervision of
Dr. Sajid Ur Rehman, Chief Scientist, Agricultural Biotechnology Research Institute, AARI
Faisalabad.

I express my heartiest gratitude to my supervisor M. Waqas Jamil , senior scientist, ABRI,

Faisalabad for his kind support, inspirational and motivational guidance and rousing exhilarating
supervision.I am indebted to pay my heartiest thanks to my parents, who always raise their hands
in prayers for me and my brother for his inspiring encouragement and guidance.

Sumraiz Ashraf

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 Table of contents

Sr. No. TOPICS

Introduction ofAyub Agricultural Research Institute

1

2 Agricultural Biotechnology Research Institute, Faisalabad.

3 Wheat Selfing & Crossing

4 Wheat data recording

5 Wheat Tissue culture

6 Sugarcane Tissue culture

7 DNA Extraction, DNA Quantification, DNA dilution

8 PCR & Gel Electrophoresis

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 AYUB AGRICULTURE, RESEARCH INSTITITE, FAISALABAD

Ayub Agriculture Research Institute (AARI) was established in 1967 on the bifurcation of
Punjab agricultural college into separate teaching and research establishments. The institute has
undergone evolution to built-up human capabilities, which has play a vital role in boosting
research efforts to meet the needs of increasing population and accelerated industrial needs and
serve farming community. AARI mission is to evolve new varieties and to develop the
technologies for food safety, food security and sustainable generation of exportable surplus,
value addition and conservation of natural resources.

Agricultural Biotechnology Research Institute (ABRI):

Biotechnology is a collection of scientific techniques used to make or modify a product to
improve plants, animals and microorganisms. Based on an understanding of DNA, scientists
have developed solutions to increase agricultural productivity.

Biotechnology:
The manipulation of the living system for the improvement of crops and livestock is termed as
biotechnology. The application of biotechnology in agriculture has resulted in benefits to
farmers, producers, and consumers.

It has an edge over traditional methods in that:

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  It has unlimited gene pool.
 More precise.
 Delivers the product in shorter time.

At the international level, genetically modified crops resistant to herbicides, diseases, insects
and pests have been produced in soybean, cotton, maize, tomato, rice and many other crops.
Some of the products are being marketed and have revolutionized the agriculture sector. The
creation of new genotypes by classical plant breeding largely depends on mechanisms that
occur during meiosis in germ cells. In contrast, modern biotechnological methods bypass the
generative cycle and produce new genotypes by introducing desired genes directly into
somatic cells. Furthermore, the development of tissue culture and molecular biological
procedures for the exchange of DNA between unrelated organisms have enabled novel genes
derived from organisms outside the plant kingdom to be introduced into recipient plants. In
addition, this modification of plant genomes by the integration and expression of foreign
DNA (genetic engineering) leads to the formation of new genotypes (transgenic plants) that
can be developed further by classical plant breeding methods.

Agricultural biotechnology mainly includes the following techniques:

1) Genetic Engineering

2) Tissue Culture

3) Genomics

Objectives:
 Genetic engineering and GMOs testing
 DNA fingerprinting and Molecular breeding
 Micropropagation and Double haploidy
 Somaclonal variation and Somatic hybridization
 Microbiology and Biofertilizer
 Capacity Building in field of Agri Biotechnology

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 Selfing and crossing in wheat
Appratus:
1) Selfing:
Wheat is a self-pollinating crop, meaning that it has both male and female
reproductive structures in the same flower and can pollinate itself without the need for an
external pollinator. To ensure selfing in wheat , simply cover the desired spike with butter
paper bag to avoid foreign pollen contamination.

Here is the procedure:

1. Identify the wheat plant that we want to self-pollinate. This should be a healthy and disease-
free plant that displays the desired traits.
2. Cover the wheat spike with a butter paper bag to prevent cross-pollination with other plants.
3. Label the plant with the date of self-pollination and the name of the pollen parent.
4. Allow the seeds to develop and mature. Once they are ready, harvest them and store them in
a cool, dry place for future use.

Wheat Emasculation

Introduction:

This method is vastly used to make new combinations and exploit the heterozygosity made while
crossing The floret of the female plant is cut open to remove male parts without harming the
ovary and cover it to avoid contamination. Male plant pollens are collected and applied onto the
spike of the female with a paint brush.

Material and Method:

1. Select a suitable plant at the suitable stage about one half in the flag leaf. Make incision on the
flag leaf to start emasculation.

2. With the help of forcep remove the middle floret so others become visible.

3. With the help of scissors cut about 3/4 portion of the florets so that the pollen become visible
to the eye.

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4. With the help of forcep remove 3 pollens from each floret.

5. Cover the spike with the help butter paper bag. Label the tag and attach it to the plant.

For pollination use a paint brush to sprinkle pollens onto the florets collected from desired male
plant.

Roguing of Wheat:
Removal of unfavourable plants from field. Agriculture, roguing refers to the act of identifying
and removing plants with undesirable characteristics from agricultural fields. Rogues are
removed from the fields to preserve the quality of the crop being grown. That is, the plants being
removed may be diseased, be of an unwanted variety, or undesirable for other reasons,

Selection Of Wheat Plant For Shorter Height And Less Disease:

The importance of phenotypic selection can not be forgotten when you are talking about the any
work related to agricultural plant improvement. As the short statured plant have more yield and
less lodging and as this year yellow and brown rust damage the yield that was the reason we
were scouting for those traits.

Selection in F1 (First Filial Generation):

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There were 120 lines of F, each from a different cross. All the plants in a line were uniform. Four
lines were missed because of no germination. Selection was made on the basis of desirable
characters i.e. Leaf and Stripe rust resistance, lodging resistance, medium height, healthy spikes,
early maturity and profuse tillerin

Selection in F2 (Second Filial Generation):

There was a total of 94 genotypes in F₂ each consisting of 6 lines. Different lines were rejected
because of heavy aphid affect, leaf and stripe rust, tall stature, lodging, late maturity and
uniformity. Uniform lines inF2 were rejected because in F₂ we get maximum segregation and
these uniform lines shows that they were not the result of crossing of selected parents in CBs but
selfing of the female parent.

Selection in F3 (Third Filial Generation):

There was a total of 120 genotypes in F3 each with2 lines. Forty lines of total genotypes were
rejected on the basis of undesirable characters i.e. late maturity, leaf rust and yellow rust
susceptibility, lodging and long stature. In remaining genotypes single head selection of desirable
plants was performed and the selected heads were tagged.

Selection in F4 (Fourth Filial Generation):

There was a total of 53 genotypes in F, each with 3 lines. Different genotypes were rejected
based on undesirable characters. Eight of total lines were tagged for CFT (cut for trial) because
of their homogeneity (early homozygosity). The remaining genotypes single head selection of
desirable plants was performed, and the selected heads were tagged.

Selection in Fs (Fifth Filial Generation):

There was a total of 90 genotypes in F, each with 3 lines. Twelve of total genotypes were
rejected because of undesirable characters. Five of total lines were tagged for CFT because of
their uniformity in appearance. In remaining genotypes single head selection of desirable plants
was performed and the selected heads were tagged.

CFT selection in F6 (Sixth Filial Generation):

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There was a total of 65 genotypes in F6 each with 3 lines. Different genotypes were rejected
because of undesirable characters including non-uniformity. Twenty-three homogenous
genotypes were tagged for CFT.

Station Yield Trials:

Station yield trials are consists of Preliminary yield trial(A-trial), Regular wheat yield trial (B-
trial) and advance yield trial (C-trial).

Preliminary Yield Trial(A-Trial):

This trial is conducted to study yield performance and other morphological characters of wheat
promising lines, harvested as CFT in the previous F4, Fs and F6 generation. Two A-trials (AI
and All) were in operation. Six lines of 14 genotypes with 3 replications were present. Out of
these 14 genotypes 2 were check varieties (Galaxy-13 and Punjab-11) for yield comparing. Each
genotype comprised the area of 6m (length) x 2.46 m (width) with 6 lines. Rust disease was only
observed to see if present or not.

Regular Wheat Yield Trial (B-Trial):

The main objective of this trial is to study consistency in yield performance and other
morphological characters of wheat promising lines selected and promoted from preliminary yield
trials of previous year. Rust disease was only observed to see if present or not. Most promising
lines were then enter in Micro yield trial.

Micro Yield Trial:

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The main objective of this trials is to study yield performance and adaptability of promising
wheat strains at different locations in Punjab. Data is recorded. All of the lines are coded in these
trials.

Wheat Immature Embryo Culture

Tissue Culture:
Tissue culture is a method of biological research in which fragments of tissue from an animal or
plant are transferred to an artificial medium in which they can continue to survive and function.
Not a single medium can be suggested for all types of plants or organs, so that the details of
culture media need to be worked out from each plant separately.

Autoclaving:

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Great care must to be taken to avoid any type of contamination during tissue culture. An
autoclave is a pressure chamber used to sterilize instruments, media, distilled water,
polypropylene sheet and other supplies by subjecting them to high pressure saturated steam at
121C for around 15-20 minutes depending upon the size of the load and the contents. First the
autoclave was filled up-to the marked level with distilled water and then media or other things
which had to be sterilized was placed in it and the machine was started. The temperature
increased slowly up to the 121C and the material was sterilized for 20 minutes. After sterilization
the temperature slowly drops to 96C and autoclave was completed. After that the items were
removed from the autoclave and taken to the laminar flow cabinet.

Working with the laminar flow cabinet:

In laminar flow air is drawn through a HEPA (High-efficiency particulate arrestance) filter and
blown in a very smooth, laminar flow towards the user and hence it avoids air contamination.
The following things should keep in mind while working with laminar flow cabinet to avoid
contamination or danger of any type.

 Turn on the UV light 10 minutes before working.

 After turning off the UV light turn on blower and fluorescent light.
 Clean the laminar flow cabinet with 70% ethanol and place all the items you going to
need inside it.
 Sterilize your hands and rubber bands with 70% ethanol. Keep the arms remain in the
cabinet after sterilization of hands.
 Sterilize the mouth of culture vessel with Bunsen burner and keep the mouth of culture
vessels towards laminar air flow.
 After using the cabinet, remove all your items including cultured tubes and sterilize it
with 70% ethanol.

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Incubation room:
In incubation room, controlled environment is present. Temperature of incubation room is 25-
26C and light is 2400-4000 lux cm. Cultures are incubated in incubation room. Date and other
informations i.e. crop name, media type and concentration etc. should be mentioned on the
culture vessels. For some cultures dark condition is required, so that wrap these cultures with
newspaper and place them in a separate container. Other cultures require both light and dark
conditions. For light condition, place the cultured vessels directly in incubation room.

Preparation of Callus Media:

Sr. # Ingredients Concentration
1 MS media(Murashige and Skoog medium) 4.43 g/L
2 D-Sucrose 30 g/L
3 2,4 D(2,4-Dichlorophenoxyacetic acid) 3 mg/L
4 Phytagel 2.5g/L

Preparation of Regeneration Media:

Sr. # Ingredients Concentration
1 MS media(Murashige and Skoog medium) 4.43 g/L
2 D-Sucrose 30 g/L
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 3 Kinetin or BAP(6-Benzylaminopurine) 0.20, 0.5 &1 mg/L
4 Phytagel 2.5g/L

Preparation of Rooting Media:

Sr. # Ingredients Concentration
1 ½ MS media(Murashige and Skoog medium) 2.21 g/L
2 D-Sucrose 30 g/L
3 NAA(1-Naphthaleneacetic acid) or 0.5, 1 mg/L
IBA( Indole-butyric acid)
4 Phytagel 2.5g/L

Material Required:
Beaker, stirrer, flask, pipette, distilled water, measuring balance, hot plate.

Protocol:
 Measure MS media and sucrose on measuring balance.
 Took 1000 ml distilled water in a beaker. Split into 2 equal parts.
 Pour 500 ml distilled water in a flask and 500 ml remains in beaker.
 Add MS media and sucrose in 500 ml distilled water of beaker.
 Took 3 ml 2, 4 D with the help of pipette in 500 ml distilled water beaker.
 Stir all three chemicals continuously.
 Then check the pH of this solution with the help of pH meter.
 Its pH is between 5.7-5.8.
 If its pH is less than 5.7 than add 1 drop of NaoH and if its pH is greater than 5.8 than add
1 drop of HCL and stir it continuously to maintain the pH.
 Add 2.5 g phytogel in 500 ml distilled water of flask to solidify the solution.
 Then put it on hot plate for 15-20 min.
 When it comes to boil, mix the previous solution and this solution. Total volume of
solution is 1000 ml.
 This media while still hot pour into 100 test tubes and each test tube contains 10 ml
media.
 The test tubes were kept in iron stands.
 After pouring media, test test tubes were wrapped with newspaper and sterilized in
autoclave and then transferef to laminar flow cabinet.
 This method was adopted for preparing each one of the above mentioned medium.

Wheat
Wheat (Triticumaestivum L.) is a major cereal staple crop that belongs to the Poaceae (grass)
family. Wheat is known as the "King of Cereals." It is a South West Asian native (Turkey).
Wheat has 2n=6x=42 chromosomes and self-pollination as a pollination method. ). Wheat is

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not only a major crop for more than a third of the world's population, but also the world's
most abundant source of energy and nutrition, as well as an important staple food crop and
the world's most traded grain.

Objective:
Variation is the basis for starting any breeding program. Variation can be induced by
mutation breeding, wide crosses and introduction etc. Through somaclonal variation we can
find plants with desired characters like rust resistance or high yielding etc.

Immature Embryo Culture

Material Required:
Flask, beaker, bleach, distilled water, ethanol, filter paper, autoclave water, forcep, knife.

Sterilization of seeds:
 Spike with immature seeds of Subhani were taken from field and the seeds were
removed from spikes.
 To make 5% solution of bleach, took 5 ml bleach in a beaker with the help of
measuring cylinder and add 95 ml distilled water in it.
 Dip 150-200 seeds in this solution covered it with aluminum foil and shake it
continuously for 20-25 min.
 Turn on the UV light 10 min before working then turned it off.
 Then took the seeds in laminar flow cabinet after removing the bleach.
 Sterilize the laminar flow cabinet and hands with ethanol.
 Turn on the lamp.
 Then dip these seeds in ethanol for 1 min.
 Give three washings with autoclave water to remove bleach from it.
 Transfer these seeds on filter paper for 10-15 min to dry the moisture to avoid
infection.

Immature Embryo Culture:

 Pick one seed with autoclave forcep and mark a cut on its back side with knife to pick
embryo from seed.
 The embryo then removed and transfers it on the callus media.
 Sterilize the forcep, knife and hands again and again with ethanol.
 Pick all the embryos from seeds and transfer it on callus media
 Then wrap the mouth of test tubes with autoclave polypropylene and rubber band.
 Cover all the test tubes with newspaper and place it in dark condition for 14 days for
the formation of callus.
 After 14 days, if callus is not formed then subculture it again on callus media.
 If callus formed, then transfer it on regeneration media.

Sub-culturing for Regeneration of shoots from callus:

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 After the callus formation, the callus was sub-cultured on the regeneration media.
Infection free culture was sort out. The callus from each tube were taken on the
sterilized glass sheet in a laminar flow cabinet and was broken into 3,4 pieces. Each
piece was placed in a test tube having regeneration medium and the mouth was
covered. Tubes were kept in the plastic stands. Sub-culture the callus again and again
on regeneration media after 14 days interval until shoots were developed. Incubated it
for 1-2 months in the culture room till shoots of desired length is obtained.

Sub-culturing for Rooting of Regenerated shoots:

 After the development of shoots from callus they were sub-cultured into the rooting
medium. Infection free culture was sorted out and infected cultures were discarded. In
laminar flow cabinet the shoots were taken out from the regeneration media and were
transferred to the rooting medium with the help of forceps. After that the mouth of the
tubes were covered. Tubes were then kept in the plastic stands and incubated room in
the culture room. Sub-culture it again and again after 14 days until the roots
developed. Incubate it for 1-2 months in culture room till roots of desired length is
obtained.
Transplanting:
 Well-developed plantlets were taken out carefully from the culture tubes with the
help of forceps. Their roots were washed in running tap water to remove the clinging
medium to avoid fungal growth and they were transplanted in a disposable cups
containing peat and soil mixture with a hole in the base to remove extra water. Each
cup was covered with a polythene bag to maintain high humidity and was kept in the
incubation room. After few days the humidity was reduced gradually by making holes
in the poluythene bags to harden the plant. The hardened plants were grown under
standard greenhouse conditions and then transplanted into the field in their growing
season (November).

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 SUGARCANE

Introduction:

Sugarcane (Saccharum officinarum L.), an old energy source for human beings and more
recently is a replacement of fossil fuel for motor vehicles. The largest producers are Brazil, India,
China and Pakistan accounting more than 50% of world production. It is an important cash crop
of Pakistan. It is mainly grown for sugar and sugary production. It also forms essential item for
industries like sugar, chip board, paper, barrages, confectionery, uses in chemicals, plastics,
paints, synthetics, fiber, insecticides and detergents.

Objective:

 For the development of somaclones having high sucrose contents and resistance toward red
rot disease.

 For single character correction.

 For fast multiplication of plants during off-season.

Meristem culture:

Healthy young meristems were collected by removing the leaf sheath from field grown plants of
sugarcane varieties 375 and 93. In the laminar air flow cabinet these young meristems were
sterilized with 70% ethanol. Outer leaves of these young meristem was removed with the help of
scalpel to obtain the inner soft meristem. This soft meristem was placed on sterilized glass sheet
and a small upper and lower portion of it was discarded to avoid the chance of contamination and
the remaining portion was chopped down into small pieces (2mm). These small pieces were
transferred into the test tube containing 2,4-D MS media with the help of forceps. Mouths of the
cultured tubes were covered with autoclaved polypropylene strips and were kept in the plastic
stands. Plastic stands were wrapped with newspaper and incubated for 14 to 15 days in culture
room for callus formation. Callus was then sub-cultured on regeneration medium for the
development of shoot. Multiple shoots can be obtained by using 2.5 mg/L concentration of BAP
or Kinetin. Developed shoots were sub-cultured on rooting medium for root development.

Transplanting:

Plantlets with well-developed roots were removed from the culture medium and the roots were
gently washed under running tap water. Plantlets were then transferred to disposable cups which
contain autoclaved peat and soil mixture and were watered and kept in the incubation room.
After few days they were transferred to polythene bag with soil and put under the shade and were
transplanted into the field in their growing season (September or February-March).

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Micropropagation:

A tissue culture technique for plant propagation in which tissue is taken from a plant and grown
in a laboratory to produce plantlets that are genetically identical to the parent.

DNA extraction, DNA quantification and DNA dilution &PCR

DNA extraction is first step to study the genomics of any organism. Sample may be change in
different organism such as leaves or seeds in case of plants and blood in human.

Objectives of DNA extraction:

 Measuring genetic diversity

 Selection of diverse parents for breeding program.
 Identification of gene of interest
Required Equipment’s:

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  Water bath
 Centrifuge machine
 Incubator
 Pipette
 1.5ml Eppendorf tubes
 Autoclaved pestle mortar
 Eppendorf stands
Required Chemicals:

 liquid nitrogen
 CTAB
 Chloroform 0.6%
 Chilled Isopropanol 0.7%
 80% Ethanol
 Double distilled de-ionized water

2X CTAB

Ingredients Quantity

Tris base 12.11 g

PVP 10.0 g

EDTA 7.44 g

NaCl 81.8 g

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 CTAB 20.0 g

dH2O Volume upto 1.0 L

pH 8.00

Protocol:

 First the water bath turn on and put the 2X CTAB extraction buffer was pre-heated in it at
65 °c.
 Then the autoclaved pestle mortar were taken from drying cabinet and if not available
than pre cooled the autoclaved pestle mortar with liquid nitrogen (-196oC).
 Than grind 2 to 3 leaves of chickpea in liquid nitrogen. Add 2-3 times liquid nitrogen to
make fine powder of sample.
 Then transferred the fine powder in 1.5 ml eppendorf tube and add 500µl CTAB that was
pre heated in water bath before grinding of sample.
 The samples were incubated in water bath for 30 minutes at 65°c.
 During incubation, tubes inverted 2-3 times to mix the content.
 Then after water bath gently shake all the samples.
 Then centrifuge the samples for 10 minutes at 13000rpm.
 After centrifugation DNA floats upward in aqueous form, take DNA with pipette and add
in new eppendorf tube.
 Then add 0.6% chloroform of DNA quantity taken. Then shake by hand.
 Again centrifuge the samples for 10minutes at 13000rpm.
 Now again take the upper portion with pipette without disturbing the below layer, add in
new tubes.
 Now add 0.7% isopropanol with respect to the volume of mixture.
 Then draw out the isopropanol, DNA pellet at bottom in the transparent form.
 Then washing with ethanol, add ethanol, centrifuge for 3 minutes at 13000rpm.
 Now left for one night to vaporize the whole ethanol.
 Then pallet dissolved in 100 µl of d3H2O.

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 (A) Leaf samples (B) Powder of leaves (C) Liquid nitrogen (D) Water bath (E)
Centrifugation of samples (G) Supernatant (H) DNA pellet

DNA Quantification

In molecular biology, quantitation of nucleic acids is commonly performed to determine

the average concentrations of DNA or RNA present in a mixture, as well as their purity.

Principle:

The basic principle involved for DNA quantification is that if more concentration of
DNA, then more absorption of light and less will pass.

Quantification by Nanodrop Spectrophotometer:

Nanodrop Spectrophotometer was used to measure DNA concentration. Nanodrop

Spectrophotometer is more accurate, time saving and its practical usage is much easy.

Sample Purity:

The ratio of absorbance at 260 nm and 280 nm is used to assess the purity of DNA or
RNA. A ratio of 1.8 is generally accepted as "pure" for DNA and a ratio of above 2.0 is
generally accepted as "pure" for RNA. If the ratio is appreciably lower in either case, it
may indicate the presence of protein, phenol or other contaminants that absorb strongly at
or near 280 nm.

Protocol:

 First of all, the computer attached to the Nano Drop was turned on.

 The loading port was cleaned with a tissue paper.

 A blank reading was taken using lul of d₂H₂O.

 Again the upper and lower arm of Nano Drop was cleaned.

 2.5ul of DNA sample was placed on the port and

 the reading (ng/ul) and DNA quality was noted.

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 This procedure was repeated for every sample.

 Computer was turned off after use.

DNA Dilution
“Making of working DNA solution of required concentration from stock DNA solution is
called DNA dilution.”
This dilution is used for working purpose while stock solution is preserved for future use.
For RAPD technique we required 15ng/ul DNA concentration and in SSR technique,
30ng/ul DNA concentration is required.

Purpose of making DNA dilution:

 It limits the need of extraction again and again.

 DNA dilution is required for applying marker techniques e.g. in RAPD & SSRs,
we require a DNA solution having known DNA strength.

Procedure:

After acquiring DNA quantification values, following formula was used to make 30ng/ul
dilution for 200ul for application of SSR markers.
C1V1=C2V2
Where,

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 C1= Known concentration of stock DNA solution (recorded from Nanodrop
spectrophotometer in ng/ul for each sample)
V1= Required volume of stock DNA for making DNA dilution (ul)
C2= Required concentration of working DNA solution = 30ng/ul (for SSR marker)
V2= Required volume of working DNA solution

Polymerase Chain Reaction (PCR)

The polymerase chain reaction (PCR) or thermocyclers is a laboratory apparatus to make
multiple copies of a sequence of DNA. This technique was developed by Kary Mullis.

Applications of PCR

 Identification of genetic finger printing

 The analysis of ancient DNA from fossils
 The diagnosis of hereditary diseases
 Detect the presence of transgene
 Mapping the human and other species genome
 DNA sequencing can also be assisted by PCR
 DNA cloning

PCR Green Master Mix (GMM)

Reagents Volume
d3H2O 10.3 ul
10X PCR buffer 2.0 ul
MgCl2 1.5 ul
dNTPs 2 ul
Primers (reverse & forward) 2 ul
Taq polymerase 0.2 ul
DNA 2 ul

Protocol:

 First of all reagent were taken out from the freezer needed for Master Mix (MM)
formation, except Taq polymerase and thawed along with DNA.
 Following the master mix template sheet (given above), the total amount of each
component which is required for the total number of samples was calculated.
 After thawing, master mix was prepared in a 1.5ml eppendorf tube by adding correct
amount of each component in the given sequence: d 3H2O, 10X PCR buffer, dNTPs, Mg
Cl2 and primers.

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  Then Taq polymerase was taken out from freezer and added into the 1.5ml eppendorf
tube to make master mix. Taq polymerase then place back into freezer.
 Master Mix was then mixed in the vortex mixer (spinner) for 2-3 seconds.
 Then 15ul of master mix was placed in each labeled PCR tubes with the help of
micropipette.
 3ul DNA sample of different genotypes was placed in thermal cycler and the reaction was
started for 35 cycles.

Steps of PCR:

1. Denaturation
In this step, double stranded DNA molecules were heated up to 94 oC which separates or
denatures them so that they become single stranded. These single strands of DNA then become
the template for the new DNA strands to be made.
2. Annealing
In order for a Taq Polymerase to start building a new strand of DNA, a primer was needed. A
primer is a short piece of DNA which will anneal to the DNA template strand where it finds a
sequence complementary to own sequence. The reaction temperature is lowered to 45–60 °C for
30–60 seconds allowing annealing of the primers to the single-stranded DNA template.
3. Extension
During this step, the TaqPolymerase will extend the primer by bringing complementary
nucleotides as it moves along the template strand. The Taq Polymerase works best at temperature
between 72-75oC so the temperature is raised so that enzyme can work efficiently. The extension
time depends both on the DNA polymerase used and on the length of the DNA fragment to be
amplified. These steps are repeated from 28-35 times. Since the reaction is essentially
exponential and since each cycle is about 5 minutes, a large quantity of DNA can be produced
for analysis in as little as several hours.

(A) PCR Machine (B) DNA Samples (C) Gradient PCR cycle profile (D) Thermal PCR cycle
profile

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 Reagents used in one reaction of PCR

Reagents Concentration Volume

Primers 20-30 ng/ul 1.0 ul

MgCl2 25 Mm 1.6 ul

dNTPs 2-2.5 Mm 2 ul

DNA Polymerase 5 units/ul 0.2 ul

PCR Buffer 10 X 2 ul

DNA sample 15 ng/ul 2.5 ul

Functions of PCR Reagents

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 Reagents Functions

Primers To initiate amplification of DNA strand

Buffer It maintains Ph.
Proper chemical environment is provided by buffer.

D3H2O It provides neutral medium for the reaction

MgCl2 It maintains ionic balance

Its’ appropriate concentration makes bands visible on gel.

dNTPs These provide building blocks to DNA polymerase for new DNA formation

Taq Polymerase It is used to synthesize the DNA copy of the specific region to be
amplified.

Basic PCR Profile

Steps Temperature Time No. of Cycles

Initial 94OC 5 min 1

Denaturation

Denaturation 94OC 30 sec

Annealing Variable 30 sec 35

Extension (1 min 72OC 1 min

per Kb
amplification)

Final Extension 72OC 7 min 1

Hold 4OC Until turned off

Gel Electrophoresis
Gel electrophoresis is a method for separation and analysis of macromolecules (DNA, RNA and
protein) and their fragments, based on their size and charge.

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Principle:

Fragments of linear DNA migrate through agarose gels with a mobility that is inversely
proportional to the log10 of their molecular weight. Smaller particles move faster and move
farther than larger one. Negatively charged particles move toward positive electrode and vice
versa. High charged particles are also more electrical field responsive.

Reagents required for gel electrophoresis:

 1X buffer
 Agarose gel
 Ethidium bromide
 DNA ladder
 Loading dye

Agarose gel:

Agarose is a dry white powder made from seaweed. It dissolves in near-boiling water, and forms
a gel when it cools. It is composed of long unbranched chains of uncharged carbohydrate without
cross links having large pores allowing for separation of macromolecules.

Electrophoresis Buffer:

There are two types of buffer use in electrophoresis i.e. TAE (tris acetate EDTA) and TBE (tris
borate EDTA). These two buffers have different ionic strength. Buffers not only establish the pH
but also provide ions to support conductivity.

TBE Buffer (10X for 1000ml) in d3H2O

Ingredients Quantity
Tris base 54.4 g
Boric acid 27.5 g
0.5 mM EDTA 20 ml

Preparation method of buffer:

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500 ml of d3H2O was taken in a beaker and all the above mentioned ingredients were placed in
it. After that the total volume was made upto 1000 ml d3H2O. The beaker was placed on the
magnetic stirrer and contents were mixed till the solution become clear.

Ethidium bromide:

Ethidium bromide binds strongly to DNA by intercalating between the bases and fluorescence a
red-orange color under ultraviolet light.

DNA ladder:

DNA and protein standards are used in electrophoresis to provide guide to the length or
molecular weight of DNA strand or protein molecule respectively. In gel electrophoresis, 100pb
ladder is used in order to determine size of unknown samples.

Loading dye:

Two types of dyes are used in gel electrophoresis.

o Sucrose dye
o 6X bromophenol dye

The purpose of loading dye is to give color to DNA sample, which helps to observe the
movement of DNA molecule in agarose gel.

For 2% small agarose gel

Reagents Quantity
0.5X TBE buffer 40 ml
Agarose gel 0.8 g
Ethidium bromide 2 ul

For 2% medium agarose gel

Reagents Quantity
0.5X TBE 200 ml
Agarose gel 4g
Ethidium bromide 4 ul

For 2% large agarose gel

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 Reagents Quantity
0.5X TBE 350 ml
Agarose gel 7/8 g
Ethidium bromide 8 ul

Protocol:

 Electrophoresis assembly
 First of all flask, comb and stoppers were washed under tap water.
 Bench area was cleaned with newspaper.
 Already present buffer was removed from electrophoresis tank washed and cleaned with
a tissue paper.
 Stoppers were fixed on gel casting tray.

Gel preparation:

 Required quantity of 1X TBE buffer was taken in a flask.

 Then weighted amount of agarose was taken and spread on the surface of TBE buffer.
 The flask was placed in microwave oven and heated till it become transparent.
 The solution was then cooled down under tap water while continuous stirring.
 Ethidium bromide was then added with the help of micropipette according to the size of
gel and solution was stirred to dissolve it.
 The gel was then poured in gel casting tray.
 The desired size comb was placed from one side of the gel. In small agarose gel 1 comb
but in medium agarose gel 4 combs can be placed.
 After that, the gel was left to solidify in tray for half an hour at room temperature.

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Loading and running of DNA sample in gel:

 When the gel became solid, then combs were carefully removed along with stoppers.
 Gel along with casting tray was placed in the electrophoresis tank such that the wells
were oriented towards the negative electrode.
 The tank was filled with enough 1X TBE buffer to submerge the gel.
 3 ul of 6X bromophenol dye was added in each PCR tube containing DNA sample with
the help of micropipette.
 10 ul of stained DNA was loaded in each well of the gel along with 5 ul DNA ladder in
the first well of each lane for comparison.
 The lid was then placed back on the electrophoresis tank and the electrodes were
connected to the power supply (black to black and red to red).
 The power supply was turned on and a constant voltage of 90 V was applied for one and
ahalf hour till the gel is completely run.

. Bands Visualization:

After the separation of DNA segments, the gel was placed in Gel Documentation System (GDS)
and observed under UV light to see the bands. The photograph of the gel was taken and saved in
the computer.

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Result:

The primer was s polymorphic as it is giving different bands in different genotypes but the
agarose gel electrophoresis is unable to differentiate the bands. So PAGE should be run to see
further polymorphism..

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