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IDENTICAL MINOR CHANGES PARAPHRASED

Internship Report

Submitted By
Abida Rafi Ullah
Internship Report
For fulfillment of the requirements the degree of
Bachelors in Biotechnology
Department of Biotechnology
Virtual University of Pakistan
Session: 2019 to 2024
Duration:
Internship Completion Certificate attached here

Introduction of Lab Organization

Services Institute of Medical Sciences Services Hospital, Lahore
Services hospital is a hospital at Governmental Level. It was built in 1958 as a separate OPD department at
Mayo Hospital. Later on it was converted into 55 bedded hospital and named as “Wahadat Hospital”. Later
on it was named as “Services Hospital, Lahore” Now it is considered in the list of biggest hospitals in
Lahore.
There are 4 labs in Services Hospital
Medical College Lab
Emergency Lab
SIMS Diagnostic Center
OPD Lab
Departments in these Labs
Hematology
Microbiology
Biochemistry
Molecular Biology
Serology
Chemical Pathology
Histopathology
Blood Bank
I have done my internship at “SIMS Diagnostic Center”.
Dedication
I want to dedicate this struggle first of all to Allah almighty and then to my Parents and all most respected
teachers who guided me in every moment of my graduation career.
All the staff of Science department were helpful and cooperative throughout the Biotechnology course.
I pay my regards to all the teaching and managing staff of Lab as well as Virtual university to allow me to
gain experiences and learning skills through my education career.

Executive Summary
I did my internship in Hematology, Biochemistry Serology, and Micro lab.
I was not to be gotten/allowed chance to do tests at Molecular lab. But I learned Molecular tests in last
semesters which I would also mention them here also.
Goals
I expect it have the practical knowledge in my career and would want to have the progress in my career.
All the learning experience was grateful and I would thanks to all the teaching staff.
May Allah Almighty bless them and may I would have a top progress in my practical ahead life.
Jazak Allah!
Table of Contents
Sr.No.
Tests
Page no.
1
CBC
8
2
Reticulate Count
9
3
Hepatitis B and C
10
4
Pregnancy Tests
12
5
RFT
13
6
Widal Test
14
7
Gram Staining
17
8
ZN Staining
19
9
Culturing of Bacteria
21
10
Urine culture and sensitivity test
23
11
Blood Culture
25
12
Semen Analysis
27
13
DNA Extraction
31
14
PCR
33
15
Gel Electrophoresis
37
16
Bibliography
40
Hematology Lab Tests
CBC Test
Introduction
The amount of each type of cell in the blood is measured by the complete blood count test. Additionally, it
offers some useful information on additional characteristics of each kind of blood cell.
Five cubic milliliters of blood must be drawn from a vein in order to perform a complete blood count test.
The sample is often taken from a vein that is visible through the skin.
Procedure
First of all, I pressed power button to start the machine.
Then Menu appeared and then I clicked the Measure.
Blood was passed between 2 electrodes through an aperture.
The analyzer separated the components of blood.

CBC machine
Observation of Patient test report
Test
Results
Reference Values
Hemoglobin
12.6 mg/dL
Male: 14-18gm/dL
Female: 12-18 gm/dL
Red cell count
3.76(10^6/uL
Male: 4.5-6.3
Female: 3.5-5.5
Hematocrit (P.C.V)
35.7%
Male: 38.3-48.6
Female: 35.5-44.9
M.C.V
94.9 Fl
83-101
M.C.H
33.5 PG
26-32
TLC
11.17 (10^3/UL
4.0- 11.0
Neutrophils
52%
50-70%
Lymphocytes
29%
20-40%
Monocytes
07%
02-08%
Eosinophils
12%
01-04%
Platelets count
338(10^3/UL)
100-400(10^3/UL)
ESR
07 mm/hour
Male: 0-9 mm/hour
Female:0-15 mm/hour
CBC Results
A "reference range" and outcomes were the two columns I noticed when I received the report. Our results
are regarded as normal if they fall within the reference range. Our results are abnormal if they deviate
from the reference range in either direction. One of the most frequent causes of our findings being
incorrect is mild anemia.
Reticulocyte Count
Introduction
Reticulocytes are immature erythrocytes. They are alternatively referred to as immature erythrocytes.
Reticulocytes are produced in the bone marrow and then released into the circulation. Approximately 48
hours after their formation, they undergo maturation and transform into fully developed red blood cells.
The function of these red blood cells is to transport oxygen from the lungs to all cells in the body.
A reticulocyte count, often known as a retic count, quantifies the quantity of reticulocytes present in the
bloodstream. Abnormal counts, whether excessively high or low, might indicate significant health issues
such as anemia and conditions affecting the bone marrow, liver, and kidneys.
Procedure
1. I placed 2-3 drops of dye solution into a test tube.
2. I added 2-4 drops of thoroughly mixed blood sample and stirred.
3. I sealed the tube and placed it in an incubator set at a temperature of 37 degrees Celsius for a duration
of 10 to 15 minutes.
4. After the period of incubation, I thoroughly combined the components and created a thin layer of dyed
blood.
5. After the films had dried, I inspected them without applying any fixing or counterstain.
6. I conducted a count of 1000 red blood cells (RBCs) and recorded the quantity of reticulocytes present.
Reticulocytes will have a reticulum or network that is dark-blue in color.
Results
If the results indicate an elevated level of reticulocytes (reticulocytosis), it might indicate the presence of
hemolytic anemia, a kind of anemia characterized by the rapid destruction of red blood cells exceeding
the rate at which the bone marrow can produce new ones.
The infant may be suffering from hemolytic disease of the newborn, a disorder that impairs the baby's
blood's capacity to transport oxygen to organs and tissues.
Hepatitis B and C Test (Rapid Test)
One finds out whether someone has ever been infected with the hepatitis C virus by use of a blood test
known as an HCV antibody test. Sometimes known as the anti-HCV test, the HCV antibody test analyses
blood for hepatitis C virus antibodies.
Apparatus
Gel Vial
Sample
Hepatitis Kit (HCV Rapid test) (HBsAG Rapid test)
Buffer
Disinfectants
Timer
Cotton swab
Spreading
Blood transfusion
Sexual intercourse
Drugs addition
Procedure
1. Before use, all the chemicals and the sample were brought to room temperature.
2. Using a dropper, I put one drop, or 10 uL, of serum into the sample well "S."
3. I put two drops of sample running buffer in the same well (S).
4. The results were seen in 5 to 20 minutes.

Results
Negative
A red/ Purple colored band appears in the control line region C
Positive
A red/ Purple colored appears in control region C as well as in the test line region T. The sample is positive
for HCV
Invalid
No bands appears in the control region
Hepatitis B and C
Pregnancy tests
Tests for determining pregnancy Examine urine or blood samples for the presence of human chorionic
gonadotropin (hCG), a hormone. Typically, two tests are conducted.
Blood test
Urine test
Blood tests
A quantitative hCG test, also known as beta hCG, determines the precise concentration of hCG in your
bloodstream. It has the ability to detect even extremely small amounts of hCG.
Urine tests
Home pregnancy tests provide the advantages of privacy and convenience, while also being rapid and
user-friendly. They are quite precise provided one adheres to the instructions. All of these pregnancy tests
function in a comparable manner. One can test urine in any of the following methods:
• Position the test stick in the flow of urine.
• Collecting urine in a cup and submerge the test stick in it.
• Collect urine in a cup and transfer it to another container using a dropper.

Renal/Kidney Function Test (RFT)

Introduction
Kidney function tests assess the efficiency of your kidneys. The majority of these tests assess the
efficiency of your kidneys in eliminating waste from your body. A kidney examination may include a
hematological analysis, a 24-hour collection of urine, or a combination of both.
Purpose
It shows
Patients who have Diabetes
Patients who have hypertension problem
Any kidney problem
Tests included in RFT
Blood Tests
Serum Urea
BUN (Blood Urea Nitrogen)
Serum Creatinine
GFR (Glomerular Filteration Test)
Serum Uric acid
Sodium
Potassium
Chloride
Urine Tests
Urine Analysis
Albumin
24 hour’s Creatinine clearance
24 hour’s Urinary Protein
Insulin clearance test
Sodium
Potassium
Chloride
Procedure
Sample Collection for Blood Tests
1. I transferred 3 to 5 ml of blood into a gel-top tube.
2. A sample was spun in a centrifuge, and blood was divided to be used.
3. Serum was put through high-tech equipment and effects were seen.

Widal Test
The Widal test evaluates if people who might have typhoid fever have antibodies against LPS and flagella
in their blood that can bind to S. Typhi cells.
It was created in 1896 and called after its creator, Georges-Fernand Widal. The Widal test is an indirect
agglutination test for enteric fever or undulant fever that looks for bacteria that cause typhoid fever.
Our intestines are sick with it. This tool is used to find salmonella.
It is used to find out if someone has enteric fever, typhoid fever, or para typhoid fever.
Causes
Caused by Salmonella typhi
Salmonella para typhi A and B
Principle
1. When patient serum is combined and incubated with colorful, smooth, and attenuated antigen
suspensions.
2. The patient's blood contains antibodies that specifically target salmonella bacteria. When these
antibodies come into contact with solutions of salmonella antigens, they cause the bacteria to clump
together, a process known as agglutination.
Agglutination Anti-salmonella antibodies
Positive Present
Negative Absence
Reagents
Salmonella typhi O antigen suspension
Salmonella typhi H antigen suspension
Salmonella para typhi A-H antigen suspension
Salmonella typhi B-H antigen suspension
Glass light with six reaction circles
Incubator
Timer
Pipettes and droppers
Vidal tubes
Precautions
1. Obtain a blood sample of 2 ml using a vacutainer with a simple red cover.
2. Avoid using samples that are haemolysed or turbid.
3. It is recommended to use serum that has been recently collected.
4. Samples may be held at a temperature range of 2-8 degrees Celsius for a maximum of 72 hours while
conducting delayed testing.
Slide Method
1. I applied a single droplet of positive control onto one of the response circles on the slide.
2. I dispensed a single droplet of isotonic saline onto the adjacent response circle. Negative control.
3. I transferred a single droplet of the patient's serum from the test tube onto the four remaining
response circles using a pipette.
4. I introduced a single droplet of Widal TEST antigen suspension 'H' into the first two response rings.
I added a single drop of each 'O', 'H', 'AH', and 'BH' antigens to the four remaining reaction circles.
6. I evenly distributed the contents of each circle around the whole circle using individual mixing sticks.
7. I carefully moved the slide in a rocking motion and checked for visible clumping within one minute.
Results Interpretation
1. Agglutination is a confirmation of a positive test result. If the positive response is obtained using 20 ul
of the test sample, it shows the existence of clinically relevant quantities of the matching antibody in the
patient's blood.
2. A negative test result for agglutination suggests that there are no clinically relevant amounts of the
associated antibody present in the patient's blood.
Micro Lab Tests
Gram Staining
Introduction
The Gram staining method was devised by Christian Gram in 1884 and subsequently refined by Hucker in
1921.The Gram's staining test is used to categorize bacteria into two groups: Gram-positive and Gram-
negative bacteria. This test aids in the categorization and distinction of microorganisms. The Gram's
staining technique categorizes bacteria into two distinct groups:

Gram-positive bacteria, which retain the main pigment Crystal violet.

Two Gram-negative bacteria that often exhibit the color of the counter stain, commonly Safranin.
Reagents for Gram’s Staining.
Primary stain:
Crystal violet 2 g

3. Ammonium Oxalate 0.8 g

4. Distilled water. 100 mL
Gram’s iodine:
1. Potassium iodide 2 g
2. Iodine crystals 1 g
3. distilled water. 100 mL
Decolourizer:
1. Acetone 50 mL
2. ethanol 50mL
Counter stain:
1. Safranin 4.0 g
2.Ethanol 95% 200 mL
3.Ddistilled water 800 mL
Procedure
1. I prepped and mounted the material onto the microscope slide prior to staining.
2. Next, I applied crystal violet, the principal stain, on the smear for a duration of 20 seconds.
3. Next, I carefully washed out the discoloration with water.
4. Next, I applied Gram's iodine, which acts as a mordant, to the smear and let it on for 1 minute.
5. Subsequently, I decanted the surplus of Gram's iodine.
6. Next, I applied the acid-alcohol decolorizer to the smear until the solution became transparent.
7. Subsequently, I carefully washed with water.
Next, I applied Safranin, the secondary or counter stain, on the smear for a duration of 20 seconds.
9. Subsequently, I delicately washed away the discoloration with water.
10. Next, I dried the surface by absorbing the moisture with bibulous paper.
Results
Gram-positive: Blue/Purple colour
Gram-Negative: Red/Pink Color
Examples
Gram-Positive: Streptococcus, Staphylococcus etc.
Gram-Negative: E. coli, Salmonella Typhi etc.

Ziehl-Neelsen Staining (ZN Staining)

Introduction
The differential staining methods were first established by Ziehl and subsequently refined by Neelsen.
Thus, this procedure is often referred to as Ziehl-Neelsen staining. In 1883, Neelsen used Ziehl's carbol-
fuchsin stain and applied heat, followed by decolorization with acid alcohol and counterstaining with
methylene blue. Therefore, the Ziehl-Neelsen staining procedures were created.
The primary objective of this staining technique is to distinguish bacteria into two categories: acid-fast and
non-acid-fast groups. Acid-fast staining is used to see bacteria, namely those belonging to the genus
Mycobacterium, that cannot be stained by simple or Gram staining methods and are resistant to them.
Regeants for Ziehl-Neelson Staining
1.Filtered Carbol Fuchsin Stain.
2.Acid Alcohol 3% v/v (20% Sulphuric Acid).
3.Malachite Green 5g/l(.05%w/v) or Methylene Blue 5g/l
Procedure
1. I started by uniformly covering the whole slide with sputum.
2. I then set the slide to dry. The smeared area need to face upwards. I let it air dry for half an hour.
3. I then heated the dry smear and repaired it.
4. Next, I used Carbol fuchsin stain to conceal the stain.
5. Next, I warmed up the smear. I used 60C for heating. Additional stain may be applied as needed. Five
minutes should pass while the hot stain is left on the slide.
6. After that, I used distilled water to wash the discoloration.
7. Next, I applied 3% v/v acid alcohol or 20% sulfuric acid to the smear, leaving it on for two to five
minutes. The smudge loses its color.
8. After that, I used distilled water to wash the smear.
9. After that, I used malachite green stain on the stain and let it on for a minute or two.
10. I then used clean water to wash the discoloration.
11. After that, I cleaned the slide's back and set it on a draining rack so the smear could dry.
12. Next, I used a microscope to look at the smear. My oil immersion goal was 100X.
Results
Reagent. Acid Fast. Non Acid Fast
CarbolFuchsin Red(Hot Pink) Red(Hot Pink)
With heat
Acid Alcohol Red Colourless
Methyle Blue / Red Blue/Green
Malachite Green

Culturing of Bacteria
Culture Media
Culture medium contains essential nutrients and physical growth characteristics that are required for the
movement and development of organisms. Not all microorganisms are capable of growing in a single
culture media. Various bacteria have distinct needs for development. There are three kinds of culture
medium used for microorganisms, categorized according to their consistency. These are
1. Solid medium.
2. Semisolid medium
3. Liquid medium (broth).
General Purpose media/Basic Media
This medium is suitable for the majority of non-demanding microorganisms. Peptone water, nutrient
broth, and nutrient agar are classified as basal media.
These media are often used for the first isolation of microorganisms.

Nutrient Agar
Ingredients for nutrient agar
I Added 10 grams of NaCl
I added 10 grams of Tryptone
I added 5 grams of Yeast Extract
I added 5 grams of Agar
In the end I added 1 liter of Distilled Water
Enriched Medium(Added growth factors)
By incorporating additional nutrients like as blood, serum, and egg yolk into the basic medium, it is
transformed into an enriched medium. Enriched medium supports the development of microorganisms
that have specific nutritional requirements. Chocolate agar, blood agar, and Loeffler's serum slope are all
examples of enriched media. Blood agar may be prepared by adding 5-10% (by volume) to a blood agar
base. Lysed agar, also known as chocolate agar or heated blood agar, is a kind of agar that has been
treated to release the nutrients from the blood cells.
Composition of blood agar
I added 0.5% peptone
I added 0.3% Beef extract/yeast extract
I added 1.5% Agar
I added 0.5% NaCl
I added Distilled Water
(Since Blood Agar is made from Nutrient Agar, above is the composition of Nutrient Agar)
I added 5% Sheep blood
pH should be from 7.2 to 7.6 (7.4)
Blood Agar
Urine culture and sensitivity test
A urine culture test may detect the presence of bacteria or yeast responsible for a urinary tract infection
(UTI). An antibiotic sensitivity test may determine the most effective antibiotic for killing a certain strain of
bacteria as they reproduce.
Procedure
Performing a colony count on pee
1. Get a clean wire loop
2. Take a microliter of urine. 3. Spread the urine out from the middle of the plate and then hit it from side
to side to level out the organisms on it.
4. Put the plate in a warm place (37 degrees Celsius) for 24 to 48 hours.

5. After the plate was kept at 37 degrees Celsius for 24 to 48 hours, count each cell on the plate.
Each colony is made up of one bacteria, which is known as a colony-forming unit.
Kirby Bauer Sensitivity test
1. I used a clean swab to pick out a single cell.
2. To put the grass on Mueller-Hinton agar, I spread it out evenly to cover the whole plate.
3. I spread the germs again in a straight line
4. I took an antibiotic disc and put it in the middle of the mueller hinton agar that had been infected.
5. I used a clean cotton swab to gently tap the disk on the agar to stick it there.
6. I kept the plates in the oven upside down at 37 degrees Celsius for another 24 to 48 hours.
Results
If bacteria would have grown we can see clearing zone of inhibition
The bacteria only susceptible to some kinds of bacteria

Blood Culture
Purpose
To be sure of bacteremia
For bacteremia and septicemia to be found
To be sure of a fungal infection
Principle
Pathogenic microorganisms in the blood thrive when fed with a specialized and nutrient-rich culture
medium, and may be primarily identified from the resulting broth culture.
Requirements
Inoculation loop
Culture swab
Syringe 10cc
Tourniquet
Alcohol pad
Gloves
Incubator
Autoclave machine
Safety hood for media pouring
Culture bottles
Nutrient Agar
Blood Agar
Chocolate Agar
McConkey Agar
S.S Agar
Procedure
1. During the process of collecting blood, I made sure to wear gloves and make use of sterilized needles
and disposable syringes.
2. In preparation for the collection, I used an alcohol pad to clean the skin all around the site.
3. I took ten milliliters of blood from the veins.
4. It was shortly after the collecting of the specimen that it was injected.

5. I placed the needle into the rubber liner of the bottle cap after cleaning it with spirit. I then poured 10
milliliters of blood into the bottles that contained brain heart infusion broth and thioglycollate broth,
respectively.
6. It was imperative that the blood not permitted to coagulate in the culture medium, since this would
have resulted in the germs being trapped inside the clot.
I took my time and carefully mixed the blood in the blood culture vial.
8. The blood culture was incubated at a temperature of 35-37 degrees Celsius for a maximum of seven
days, and the turbidity of the blood culture bottle was checked twice. At seven days, brucellosis is thought
to have occurred.
9. In the event that turbidity arises within seven days, a subculture may be carried out on blood agar,
McConkey agar, Chocolate agar, or S.S. agar, according on the circumstances.
Results
No turbidity means no growth
Semen Analysis
Introduction
During a sperm and sperm analysis, a man's sperm and sperm are examined. Additionally referred to as a
sperm count or male fertility test, the findings of this test reveal not only the quantity of sperm that are
discharged but also their form and the degree to which they are able to migrate.
Ejaculating during sexual activity causes males to produce a thick fluid known as sperm, which is produced
by the penis of the male. In order for sperm to fertilize an egg, it must first be transported out of a man's
body.
Procedure
Sample is collected in a cup
Sample is observed under microscope
Result of a Random Patient
Test Result Previous Results Reference Value
Sexual Abstinence 04 Days
Time of Ejaculation 11:57 AM
Physical Examination
Colour: Whitish Grey
pH 8.0
Volume 04 ml
Liquification Time 30 Mint
Viscosity Thick
Sperm Count 120.0 million / ml 20.0 million /ml
MOTILITY
After 01 hr 75 %
After 02 hrs 70 %
After 04 hrs 65 %
After 06 hrs 60 %
Forward Progression GOOD
MORPHOLOGY
Round Cells 01-02 /HPF
Pus cells NIL /HPF
RBCs NIL
Total Abnormal Spermatozoa 20 %
Heads 10 %
Middle pieces 05 %
Tails 05 %
Conclusion NORMAL SEMEN
Results
How many sperms there are (concentration).
How your sperms are moving (Motility).
What your sperms look like (Morphology).
Urine Examination
Urinalysis (Urine Test)
Urinalysis is one way to find certain illnesses in their earlier stages. They include:
Kidney disease
Liver disease
Diabetes
Urinalysis Procedure
Urine may be analyzed in three different methods, and it's possible that your test will employ all three of
them.
A visual examination is one that examines the color and clarity of the image. In the event that your urine
contains blood, it may be crimson or dark brown in color. When you have cloudy urine, it may be an
indication that you have an infection, whereas foam might be a symptom of renal damage.
In a microscopic examination, items that are too tiny to be seen in other ways are looked for. There are a
number of substances that a microscope may detect that should not be present in your urine, including
the following:
The presence of crystals, which are aggregates of minerals and may be an indication of kidney stones, as
well as red blood cells, white blood cells, bacteria, and crystals.

Dipstick testing, which is the third component of urinalysis, involves the use of a thin plastic strip that has
been treated with chemicals. If the levels are higher than usual, the chemicals on the stick will react and
change color. This is done by dipping the stick into your urine. The dipstick test may be used to check for
the following things:
• Acidity, or pH. It is possible that you have kidney stones, a urinary tract infection (UTI), or another illness
if the acid is abnormal.
It is protein. This may be an indication that your kidneys are not functioning properly. The kidneys are
responsible for ridding the blood of waste materials.
It is glucose. Diabetes may be deduced from the presence of a high sugar level.
• Cells that are white based. It is possible that these are symptoms of an infection or inflammation, which
might be occurring in the kidneys or anyplace else in the urinary system.
Urine analysis

Molecular Lab Tests

DNA EXTRACTION
The process of separating DNA from other components of blood is referred to as DNA extraction, and
DNA may be used for the purpose of isolating, identifying, and extracting the gene that is desired.
PRINCIPLE:
DNA extraction involves breaking up the nucleus membrane, cell membrane, and cell wall to release
highly intact DNA into a buffer solution. This is followed by DNA precipitation and removal of substances
that might be contaminated, such as lipids, polysaccharides, and proteins.
You can get DNA from plants, animals, nails, and hair, but most of the time we get DNA from blood. The
DNA that was taken out is kept at -20.
CHEMICALS:
Distilled water
Absolute ethanol
SDS
Ethanol 70%
Buffer A and buffer B
MATERIAL:
Centrifuge tubes
Weighting balance
Chilled meter
Beakers
Ice bucket
Micro pippete
Centrifuge
Measuring cylinder
Water bath
PROCEDURE:
Take 200L and add 1mL lysis buffer.
Vortex mixing for 5 min.
Centrifuge at 13000 rpm/15 min until a while colourless pallet is formed in bottom of eppendrop.
Discard remaining solution preventing the pallet.
Add 20 L PK.
Add80 L SDS
Add 250 L buffer AI.
Vortex mixing for 5 min.
Put the sample in overnight digestion at 58 degree Celsius.
Add 300 L PCI and do vortex mixing for 5 min
Centrifuge at 13000rpm 15 min at 4degree Celsius
Take top aqueous layer and separate it into new tube.
Add same amount of isopropanol in 1:1 ratio.
Incubate at room temperature for 5 min.
Centrifuge at 13000 rmp /10 minutes at 4 degree Celsius.
Discard ethanol and put the pallet in air for drying.
Add 150-200 L water and store at -20 degree Celsius.

Human sample

Step 1

Sample 2 Thread like structure of DNA

Extracted DNA
Discussion:
Reagents
Uses
Lysis buffer
Lysis buffer is used to disrupt and degrade the plasma membrane of cell.
Proteinase K
Proteinase K is used to degrade the protein molecules present in solution
SDS
SDS is sodium dudicycle it help to neutralize
the solution because SDS have negative charge and it give negative charge to all particles present in
solution due to which DNA repell and separate.
A1
A1is also called TNE
T-Tase element control pH
N-help in charge neutralization
E-EDTA chilling agent –clumpDNA-prevent from degredation
PCI
PCI is also called phenol chloroform isoamyle alcohol .Phenol-chloroform extraction is used to separate
and also help to form two layers
Iso-proponal
Iso-propanol is helping in the visibility of DNA molecule ,when this reagent idol in solution it form layer
around DNA due to this layer DNA become in soluble in solution and become visible.
Ethanol
Ethaanol is used as washing purpose all the removing impurities soluble in ethanol and then ethanol is
precipitate act and DNA extracted.
Dd water
Add water also called double distilled water it is used to save the DNAmolecule for same time.
Result:
You have to purified DNA after using organic method.
Polymerase Chain Reaction (PCR)
Introduction
PCR Principle:
PCR is a commonly used amplification process that generates vast quantities of a particular DNA segment
using a tiny quantity of material, which may be either a target sequence or a DNA template. High
temperature causes the denaturation of double-stranded DNA into single-stranded DNA.DNA polymerase
extends the 3' end of a custom-designed oligonucleotide by adding nucleotides when it is paired with
longer template DNA.PCR utilizes the enzyme DNA polymerase to catalyze the synthesis of DNA from
deoxynucleotide substrates on a single stranded DNA template. DNA polymerase has the ability to use an
oligonucleotide as a primer and extend its 3' end, resulting in the creation of an elongated stretch of
double-stranded DNA. Therefore, if a synthetic oligonucleotide is paired with a single-stranded template
that has a region that is complementary to the oligonucleotide, it may anneal to the template.
Components of PCR:
Magnesium:
Magnesium influences the process of primer annealing and template denaturation, as well as the activity
of the enzyme. Inadequate magnesium levels result in reduced creation of desirable products, since
magnesium serves as a cofactor. Conversely, an excessive quantity of magnesium leads to the generation
of non-specific amplification products. When base pairing becomes too strong, primer annealing fails.
Magnesium facilitates DNA extraction and forms complexes with dNTPs, which serve as the real substrate
for Taq polymerase when the magnesium concentration is sufficiently high.
Nucleotides, specifically deoxynucleotide triphosphates (dNTPs):
All kinds of nucleotides are crucial for the reaction since they serve as the building blocks for new DNA
strands DNA synthesis is unable to proceed without deoxyribonucleotide triphosphates (dNTPs).The
components of the nucleic acids are Adenine (A), Guanine (G), Cytosine (C), Thymine (T), and Uracil (U).
Primer design:
Primer binding to the template is enhanced by having high specificity and strength. If the primers fail to
bind to the right template, an incorrect sequence will be amplified.
Avoid using complementary nucleotide sequences inside the primer and between primers. For PCR to be
highly specific and efficient, it is crucial to choose primer sequences that are ideal and to maintain an
acceptable primer concentration. It is preferable for the primers to terminate with a GC sequence in order
to achieve a strong attachment to the template. Avoid using primers with consecutive runs of three or
more Cytosine or Guanine nucleotides at the 3' ends, since this might lead to non-specific binding to G or
C rich regions in the genome, causing misprinting. Avoid using thymine and adenine base pairs with a
single hydrogen bond at the 3' end of primers, since this weakens the primer's binding to the template
DNA.A single or a few copies of DNA segments that are many orders of magnitude larger are replicated to
generate hundreds to millions of copies of a specific DNA sequence. PCR is a widely utilized technology in
research facilities and clinical settings for a wide range of applications.
Primers:
Primers are synthesized DNA strands that are around 18 to 25 nucleotides long and are complementary to
the 3' end of the template strand. The process of DNA synthesis is initiated by DNA polymerase at the 3'
end of the primer. Two primers, a forward primer and a reverse primer, need to be constructed for PCR.
The forward primer is located at the beginning of the gene, whereas the reverse primer is positioned
elsewhere. The reverse primer is the reverse complement of the 3' end of the gene, considering the sense
strand (5'-3') of the gene for primer design. The reverse primer is complementary to the 3' end of the
sense strand (5'-3'), whereas the forward primer is complementary to the 3' end of the antisense strand
(3'-5').
DNA polymerase:
Taq DNA polymerase is the most common DNA polymerase (Thermus aquatics a thermophilic bacterium)
because of high temperature stability. DNA
DNA/RNA template:
Each template utilizes a distinct PCR method. The segment of interest, referred to as the target sequence
DNA template, is a specific area or segment of DNA that has to be amplified. The DNA template is the
specific DNA sequence that is being targeted. Conventional PCR utilizes DNA templates, such as
complementary DNA (cDNA), genomic DNA (gDNA), or plasmid DNA. In contrast, RT-PCR employs RNA
templates. It is crucial to optimize the effectiveness of PCR by utilizing enough amounts of high-quality
DNA or RNA template.
Buffer:
KCL buffer
These are supplied with Taq polymerase.
PCR working:
PCR works in 3 major steps which are repeated for 30 or 40 cycles.
Denaturation: it is a heating step separates complementary strands of DNA by hydrogen bond breaking
and it is done by heating 94 degree Celsius for 45-60 seconds.
Annealing: At a temperature of 54 degrees Celsius, this phase involves the process of cooling the reaction
mixture after denaturation. It results in the hybridization of primers to a distinct strand of the DNA
template. The annealing temperature is determined by the melting temperature of the primers, which are
bound to the separated DNA strands. The guanine-cytosine (GC) concentration and length of the primers
should be enough to ensure stable binding with the template.
Extension: Once annealing has occurred enzyme synthesize new DNA strands. Temperature for
polymerase 40-60 seconds is done by DNA polymerase the reaction mixture is heated at 72 degree Celsius
which is ideal working. The polymerase adds nucleotide complimentary to template on 3’OH of primers
thereby extending the strand new.
Final extraction:
Repeated three steps 35-40 times to produce million of exact copies of target DNA. Several cycles
completed and maintained short term storage pf amplified and in PCR profile final extraction is run. It is
programmed at 72 degree Celsius.
Protocol:
Additionally, one positive control, one negative control, and one non-template control were included in the
response.
The prepared master mix was distributed evenly throughout all PCR tubes.
The PCR tubes were inserted into a thermocycler device, which enables the DNA amplification process to
be repeated in three fundamental stages.
This machine is equipped with a heat block that has holes for inserting PCR tubes and plates storing the
PCR reaction mixture.
The equipment systematically and precisely adjusts the temperature of the blocks in a controlled manner.
Master Mix preparation:
Reagents
Volume Required
dNTPs
2.0l
MgCl2
2.5l
Taq polymerase
1.0l
Forward primer
1.0l
Reverse primer
1.0l
NH4 buffer
4.0l
N.W
10.5l
Total volume
22l
PCR machine
Results:
Products of PCR are visualized on 2 % Agarose gel under UV trans illuminator and can be measure along
DNA ladder.
Discussion:
The optimal denaturing conditions are achieved at a temperature of 94-95 degrees Celsius for a duration
of 30-60 seconds. Prolonged duration and elevated temperature might result in a decline in enzyme
function. Insufficient cooling may lead to incomplete denaturation of the template target and PCR
products. During the annealing process, a greater GC content leads to a more robust binding. If the GC
content is lower, the duration of my binding will grow to have a stronger binding (more than hydrogen
bonding) between the template and primer. Then, the prolonged elongation phase is conducted to
guarantee that all products are double-stranded.
Agarose Gel Electrophoresis:
Principle:
DNA fragments are sorted based on their size using gel electrophoresis on a solid medium such as
agarose gel. DNA samples are transferred into designated wells using a pipette, and then an electric
current is applied. This current causes the negatively charged DNA to move towards the positively charged
anode. The pace of migration in the gel is directly related to the smaller size fragments, resulting in their
faster movement towards the bottom. The visualization of DNA fragments is achieved by incorporating an
intercalating dye, ethidium bromide, into and throughout the gel. The dye binds to the DNA fragments,
allowing them to travel across the gel.
Exposing the intercalated dye to UV light results in fluorescence, with the larger fragment exhibiting a
more intense fluorescence. The smaller pieces contain a lesser amount of DNA and consequently absorb
less dye, resulting in a less strong fluorescence. However, each fragment of the same kind of molecule is
present in equal amounts. A ladder consisting of DNA fragments of known size may be electrophoresed
concurrently with unknown fragments to determine their sizes.
Protocol:
Preparation of the Gel:
Into an rlenmeyar flask weight out the appropriate mass of agarose .Using w/v percentage solution
agarose gels are prepared using. With most gels ranging between 0.5% -2% the concentration of agarose
in gel will depend on the sizes of DNA fragments to be separated. The volume of buffer should not be
greater than 1/3 of capacity of flask.

Preparation of buffer
Add running buffer to the agarose that has flask in it. Mix by swirling. Most of the time, TAE and TBE are
used as running gel buffers.
Melt the mix of buffer and glucose. To do this, most people heat the microwave for one to three minutes,
but it can also be done over a Bunsen flame. Every 30 seconds, take the flask off the counter and shake
the contents to mix them well. Do this again and again until the agarose is gone.
Add ethidium bromide (EtBr) until the concentration is 0.5 g/ml. Instead, the gel could be dyed after
electrophoresis in running buffer with 0.5 g/ml EtBr for 15 to 30 minutes, and then the running buffer
could be used to remove the stain for the same amount of time.
If you dissolve the agarose in a water bath at 65 degrees Celsius, you can let it cool on a table.
You could also tape the open edges of the gel tray to make a model, then put the gel tray into the casting
device. To make wells, put the right tool into the gel mold.
Melt the agarose and pour it into a gel mold or casting box. Leave the agarose to set at room temperature.
Then, take off the comb and put the gel in the gel box.
Gel can also be wrapped in plastic wrap and kept until it is time to use it at 4 degrees Celsius.

Gel tank
Separation of DNA Fragments and Setting up of Gel Apparatus:
In the gel box place the agarose gel. Fill gel box with buffer until the gel is covered.
Add loading dyes to DNA samples to be separated Gel loading dye is made typically made at 6X
concentration (0.25% xylene cyanol, glycerol,0.25% bromphenol blue.
Program the supply power to desired voltage (1-5V/cm between electrodes).
To the power supply attach both electrodes of gel box. Turn on power supply and verify that both box gel
and power supply are working.
Remove the lid slowly and carefully load the DNA sample into gel. An appropriate DNA marker size should
always be loaded along with experimental samples.
To the gel box replace the lid. The cathode should be closer the wells than anode. Double check electrodes
are plugged into correct slots in supply power.
Turn on power. Run the gel until dye has migrated to distance appropriate.

Comb Rubber band

Gel tray Voltage
Bands
Results:
Pictures depict the results obtained after running PCR products via gel electrophoresis. After the process
of separation, the resultant DNA fragments are observable and exhibit distinct and well-defined bands.
The ladder or DNA fragments should be sufficiently separated to enable accurate assessment of the size
of the sample band. The DNA fragments measuring 765 bp, 880 bp, and 1022 bp are separated using a
1.5% agarose gel alongside a lengthy DNA ladder.

Discussion:
Agarose Gel:
Agarose gel the standard percentage 1% is mostly used, the % of agarose gel is depend upon the DNA
fragments .The percentage of agarose determined how the DNA separated and resolution of final gel.
Deionized water:
Deionized water is used only for cleaning, and diluting things of electrophoresis buffer. TAE buffer made
pure and deionized water.
What is TAE/TBE buffer?
The buffer facilitates the movement of ions, which in turn enables the flow of electric current through a
gel and ensures a steady pH level. The prevailing method for DNA separation is the use of TAE/TBE
buffer.TAE/TBE buffer is a solution that consists of a combination of Tri base, acetic boric acid, and
EDTA.The acetic boric acid and Tris compounds aid in maintaining the solubility of DNA in water. EDTA
protects enzymes by inhibiting DNA degradation.
How to calculate concentration:
You can dilution plan by calculating unknown quantity using this formula C1V1=C2V2 where :
Concentration 1*Volume 1=Concentration 2* Volume 2
EDTA( Ethylene –diamine –tetra-acetic):
Ethylene-diamine-tetra-acetic acid (EDTA) is a commonly used chelating compound in molecular biology.
EDTA facilitates the competition between metal ions and protons for binding. The activity of DNase, an
enzyme that cleaves the phosphodiester link of DNA, is attributed to these metal ions.
Mixing the gel;
After preparing the 0.5XTBE buffer solution, you may proceed to construct a 1% agarose gel. Once the gel
is complete, you can start mixing it.
Ethidium bromide(EtBr):
Ethidium bromide is a monomeric phenanthridine dye that demonstrates a 20 to 25-fold increase in
fluorescence when it binds to double-stranded DNA. The color intercalates between neighboring base
pairs in the double-stranded DNA. The intercalated material exhibits an increase in fluorescence when
exposed to UV excitation light. Ethidium bromide is believed to contribute, at least in part, to energy
transfer from DNA bases to the dye. The dye permeates the electrophoretic gel with high efficiency and
speed. Ethidium bromide may be precast in gels for post-staining and as a pre-stain for RNA in
formaldehyde-agarose gel electrophoresis.
Loading Dye:
The purpose of loading dye is:
To denature the sample
To make sample heavier it easy to get into pocket and stays there
To visualize how far the gel has run
Mixed with DNA sample prior to electrophoresis running a gel loading dye is colored dye .Sample buffers
for agarose gels usually heavier something water like glycerol or sucrose together with tiny amount of dye
like bromophenol blue.
DNA ladder:
A DNA ladder is a solution containing DNA molecules of varying lengths that is used in agarose or
acrylamide gel electrophoresis. In order to determine the size of an unidentified DNA molecule, it is used
as a reference and separated from other molecules based on their movement in an electric field across a
gel. The DNA molecules migrate across the gel at varying velocities. The compact shape of supercoiled
plasmid DNA allows it to flow through the gel at the quickest rate, whereas linear DNA fragments of the
same size as the open circular form move at a slower pace.
Bibliography
References
http://www.cpet.ufl.edu/wp-content/uploads/2013/10/Intro-Gel-Electrophoresis-manual.pdf
https://webfiles.uci.edu/treseder/public/Protocols/Agarose%20Gel%20Electrophoresis.pdf
https://www.med.unc.edu/timetoconceive/study-participant-resources/pregnancy-test-instructions/
End of Report
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