INTERNSHIP REPORT

A report submitted in partial fulfillment of the requirements for the Award of Degree of
BACHELOR OF SCIENCE
in
ZOOLOGY
By

K.K.SENBAGAPPRIYA

Regd. No.: 21MUZO1005

At
BHARATHIDASAN UNIVERSITY
(Duration: 22/05/2023 to 05/06/2023)

DEPARTMENT OF ZOOLOGY
MUTHAYAMMAL COLLEGE OF ARTS AND SCIENCE
(AUTONOMOUS)
Affiliated to Periyar University, Salem,
Accredited by NAAC with ‘A’ grade, Recognized by UGC under Section 2(f) & 12(B),
Rasipuram - 637 408, Namakkal District, Tamil Nadu.

JUNE 2023

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 MUTHAYAMMAL COLLEGE OF ARTS AND SCIENCE (AUTONOMOUS)
Affiliated to Periyar University, Salem
Accredited by NAAC with ‘A’ grade, Recognized by UGC under Section 2(f) & 12(B),
Rasipuram - 637 408, Namakkal District, Tamil Nadu.

DEPARTMENT OF ZOOLOGY

CERTIFICATE

This is to certify that the “Internship report” submitted by name K.K.SENBAGAPPRIYA

(Reg. No.: 21MUZO1005) is work done by Dr.S.Achiram and submitted during 2023 –
2024 academic year, in partial fulfillment of the requirements for the award of the degree of
BACHELOR OF SCIENCE IN ZOOLOGY, at BHARATHIDASAN UNIVERSITY,
TRICHY.

Internship Coordinator (College) Head of the Department

Submitted for the viva- voice examination held on__________

Internal Examiner External Examiner

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 ACKNOWLEDGEMENT

 First I would like to thank Dr. S. Achiraman, Associate Professor, Department of

Environmental Biotechnology, Bharathidasan University for giving me the
opportunity to do an internship within the organization.
 I specially thank to the Ph.D. scholars and my co-interns who trained me in chemical
ecology research lab, Department of Environmental Biotechnology, Bharathidasan
University, Trichy with the patience and openness the created an enjoyable working
environment.
 My heartfelt thanks to Mr. K.P. Ramasamy, Chairman. Mr. Muthuvel ramasamy,
Secretary, Vanetra group of Institutions, Rasipuram, to provide all necessary facilities.
 I am indebted to Dr. R. Selvakumaran, Director Academics. Dr. S.P. Vijeikumar,
Principal. Dr. A. Stellababy, Vice Principal. Dr. M.N.Periasamy, Dean
Administration and Dr. P. Gowrisankar, COE, Muthayammal College of Arts and
Science (Autonomous) Rasipuram who granted permission for doing this internship
work.
 I feel proud in expressing my gratitude to Dr. S. Suganya, Assistant Professor and
Head, Department of Zoology, Muthayammal College of Arts and Science
(Autonomous), Rasipuram, for her guidance and constant encouragement in
successful completion of this internship.
 I also thank my class in-charge and guide Dr. V. Vinita Vinjoy Jerusha, Assistant
Professor, Department of Zoology, Muthayammal College of Arts and Sciences
(Autonomous), Rasipuram for timely help and Co-ordination and parental approach.
 I also thank all my teaching and non-teaching faculty members, Department of
Zoology, Muthayammal College of Arts and Science, Rasipuram, for their kind
advice and valuable suggestions.
 My special thanks to my Parents, Family members and Friends for their kind help
and constant support.

(K.K.SENBAGAPPRIYA)

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 ABSTRACT

ANIMAL HANDLING
 Animal handling requires proper care for which only technical personnel are hired.
Institutions should employ people who are trained (pharmacologist, veterinary
scientists) for handling laboratory animals and should provide both formal as well as
some on-duty training to ensure the proper handling of animals for which certain
refresher training as well as induction programmes are run by certain animal welfare
committees in different institutes and universities. These courses are required to be
taken up by the personnel once every three years (Sikes et al., 2016).
 The surfaces with which the animal may have high physical contact should be
disinfected properly (Walraven and Duffy, 2017). Animal cages, feeding bottles, as
well as watering devices should be kept clean as well as free from any contamination.
All these consumables should be washed at least once every 2 weeks. Disinfection of
the cages should be done at a temperature of 82.2°C or higher so that a vegetative
pathogenic organism gets destroyed properly by thermal effect. Waste disposal should
also be done regularly for which incineration is the most preferred method. The waste
storage area should be kept separate; and if the waste is biological in nature, then it
should be kept in cold storage to prevent its decomposition (Brandstetter, 2016).

MOLECULAR TECHNIQUES
 The purpose of this chapter is to outline some of the common recombinant DNA
methods in use today. These techniques are usually employed to isolate a defined
portion of the genome, mostly a gene, from an organism or tissue of interest and,
thereafter, to characterize the structure and function of this genetic material. To isolate
a gene, genomic DNA is extracted from a selected tissue. For a better handling the
relatively large DNA molecules are cut into a mixture of fragments by restriction
endonucleases. The fragments are then separated from each other according to their
size by gel electrophoresis. A procedure called Southern blotting is used to verify the
presence of the desired gene in one of the DNA fragments separated on an agarose
gel. The DNA fragments are transferred from the gel to a filter whereby the original

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fragment pattern is maintained. Then, a single-stranded DNA or RNA probe specific
for the gene to be isolated is hybridized to its target fragments fixed to the filter. A
radioactive or fluorescent tag is attached to the probe for subsequent identification. In
cases where only transcribed sequences are to be isolated cytoplasmic messenger
RNA (mRNA) is prepared instead of DNA. Analysis of RNA by a technique similar
to Southern blotting is termed Northern blotting. Preservation of DNA sequences is
usually achieved by DNA cloning. DNA cloning involves the insertion of a DNA
fragment into a DNA vector and the stable incorporation of the recombinant DNA
into a suitable host. Propagation of the host facilitates the amplification of the
recombinant DNA for subsequent analysis.

ORGANIZATION INFORMATION

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SHANMUGAM ACHIRAMAN, Associate Professor, Department of
Environmental Biotechnology

CHEMICAL ECOLOGY RESEARCH LAB (CERL)

 In CERL for the past twenty years research students, postdocs and collaborators were
engaged in pheromone research. Their primary research started with identification and
characterization of mice pheromones and their role in chemical signaling (Sex
attraction). They made several discoveries like identification of male specific
pheromone and estrus (Ovulation) specific pheromone in urine and gland of rodents
(mice and rats). This pheromone technology can be used for trapping rodents and it
will be effective and pest-specific (Environment friendly).
 They are also dealing with the endocrine modulating role of
pheromones/allelochemicals in alleviating the reproductive toxic effects of pollutants
in rodent and fish model. Having gained expertise in rodent pheromone biology
research, we developed interest towards livestock production so as to use this
pheromone technology to develop a cost-effective kit for oestrus (ovulation) detection
and artificial insemination in farm animals.
 Currently, they are in the path of metagenomic approaches in faecal microbiota to
detect the reproductive status and to correlate with pheromones of farm animals. In
addition, a group of students worked on diagnosis and treatment of cancer using
nanotechnology.

CONTENT

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S.NO. TITLE PAGE No.

1. LEARNING OBJECTIVE 8

2. WORK SCHEDULE 9

PHASE I – MOLECULAR TECHNIQUES 10

3. INTRODUCTION 10

4. PROTEIN ESTIMATION 10

5. ELISA 14

6. SDS PAGE 22

7. RNA ISOLATION & QUANTIFICATION 26

8. PCR 28

9. SERUM SEPARATION 31

10. CONCLUSION 32

PHASE II – ANIMAL HANDLING 33

11. INTRODUCTION 33

12. ANIMAL CARING & HANDLING 33

13. CONCLUSION 44

14. BIBLIOGRAPHY 45

LEARNING OBJECTIVE

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 Internships are generally thought of to be reserved for college students looking to gain
experience in a particular field. However, a wide array of people can benefit from
Training.
 Internships in order to receive real world experience and develop their skills.
 An objective for this position should emphasize the skills students already possess in
the area and their interest in learning more.
 Internships are utilized in a number of different career fields, including architecture,
engineering, healthcare, economics, advertising, education and many more.
 Internship is used to allow individuals to perform scientific research.
 Specifically designed to allow people to gain first-hand experience working.
 Utilizing internships is a great way to build your resume and develop skills that can be
emphasized in students resume for future jobs.
 When they are applying for a Training Internship, make sure to highlight any special
skills or talents.
 Interns can stand apart from the rest of the applicants so that they can have an
improved chance of landing the position.
 Our internship project includes 2 phases: Molecular Techniques and Animal
Handling.
 OBJECTIVES OF MOLECULAR TECHNIQUES: It can be applied to develop
and improve drugs, vaccines, therapies and diagnostic tests that will improve human
and animal health.
 OBJECTIVES OF ANIMAL HANDLING: The proper care and use of laboratory
animals in research, testing, teaching, and production (animal use) require scientific
and professional judgment based on the animals’ needs and their intended use.

WEEKLY OVER VIEW OF INTERNSHIP ACTIVITIES

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 OR WORK DIARY

1ST WEEK

DATE DAY NAME OF THE TOPIC COMPLETED

22.05.2023 Monday General Introduction

23.05.2023 Tuesday Protein Estimation

24.05.2023 Wednesda ELISA

y

25.05.2023 Thursday Serum Separation

26.05.2023 Friday SDS PAGE

27.05.2023 Saturday Zebra Fish

2ND WEEK

DATE DAY NAME OF THE TOPIC COMPLETED

29.05.2023 Monday RNA Isolation

30.05.2023 Tuesday RNA Quantification

31.05.2023 Wednesda PCR

y

01.06.2023 Thursday Animal Handling (Rat, Mice)

02.06.2023 Friday Animal Sacrifice demo, Vaginal Cytology

03.06.2023 Saturday Animal Sacrifice (Individual)

DATE DAY NAME OF THE TOPIC COMPLETED

05.06.2023 Monday Feedback class and Certificate distribution

PHASE – I
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 [MOLECULAR TECHNIQUES]

INTRODUCTION

 Molecular techniques or molecular biology techniques are common methods used in

molecular biology, biochemistry, genetics, and biophysics which generally involve
manipulation and analysis of DNA, RNA, Protein and lipid. Some of the molecular
techniques are PCR, PAGE, Blotting techniques.

PROTEIN ESTIMATION

PROTEIN EXTRACTION

AIM
 To determine the protein extraction from given sample using RIPA [Radio Immuno
Precipitation Assay] buffer.

PRINCIPLE
 Protein is a fundamental building block of life and proteins are the workhorses within
and between cells. Protein extraction is the process of isolating and purifying protein
from the samples.
 RIPA buffer is a lysis buffer used for rapid, efficient cell lysis and solubilization of
proteins from both adherent and suspension cultured mammalian cells.

MATERIALS REQUIRED
 Given sample
 RIPA buffer
 Triton X- 100
 Tris HCl
 TCA [ Trichloroacetic acid ]

PROCEDURE

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  Harvest a sample [adipose tissue] and snap freeze in liquid nitrogen [-20 degree
Celsius or -80 degree Celsius stored]
 100mg sample + 1ml RIPA buffer with protease inhibitor and keep it on ice.
 Spin at 6000 rpm for 15 minutes at 4 degree Celsius.
 Sonication for 5 minutes at low temperature.
 Add Triton X – 100 to a final concentration of 1%
 Incubate at 4 degree Celsius for 30 – 60 minutes.
 Centrifuge at 12000 rpm for 15 minutes at 4 degree Celsius.
 Transfer the supernatant in new micro centrifuge tube.
 Store the extracted sample at -80 degree Celsius until further analysis.

PROTEIN QUANTIFICATION
The process of determining the concentration or amount of protein present in a
sample. It is important for various applications, including; Experimental Design, Sample
Normalization, Enzyme Kinetics, Protein – Protein Interactions and Biomarker Discovery.

METHODS FOR PROTEIN QUANTIFICATION

Spectrophotometric assays Bradford assay, Lowry method, BCA assay

Fluorescence – based assay Fluorescent Dye Binding Assay, Fluorescent
Antibody - Based Assays, Fluorescent Protein
–based assays
Mass Spectrometric assay MALDI-MS, ESI-MS,
SRM/MRM, MS/MS, IMS-MS

BRADFORD ASSAY
 Highly sensitive to light.
 Detect low concentration of proteins.
 Fast and straight forward method and suitable for high–throughput protein
quantification.

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BRADFORD REAGENT (1X)
CHEMICALS 250 Ml 100mL
Coomassie Brilliant Blue G250 0.25 g 0.01 g
Absolute Ethanol 12.5 ml 5ml
O – phosphoric acid 25 ml 10 ml

 Add these Bradford reagents and vortex.

 Overnight incubation.
 Compared with BSA, BGG standards.

STANDARDS
Stock solution is BSA=50mg/ml
Working solution is r2=0.99
Absorbance read at 595nm using spectrophotometer

PROCEDURE
 Add 4µl sample.
 Dilute the sample with 96µl distilled water.
 Add 2.5 ml Bradford reagent in it.
 Incubate for 2 minutes at dark room.
 Reading in spectrophotometer.

BRADFORD CALCULATION
Y = 0.0068X + 0.0233
Y – 0.0233 = 0.0068X
X = (Y – 0.0233) ÷ 0.0068
Where,
Y = OD value
X = Concentration of protein

OD value for Heart is 0.665

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 X = (0.665 – 0.0233) ÷ 0.0068
= 94.367µg/ml
Convert into 1mL:
X (94.367) = 100
X = 100 ÷ 94.367
= 1.059µg/ml
RESULT:
 Blue color shows protein presence. Intensity of blue color shows concentration of
protein.

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 ELISA [ENZYME LINKED IMMUNO SORBENT ASSAY]

AIM
 To detect and measures the antibodies in the given blood samples.

PRINCIPLE
 Based on basic immunology response.
 Lock (antibody) and key (antigen) concept – key fits into the lock.
 Enzyme conjugate substrates – bound to a secondary antibody that binds with the
antibody – antigen complex.

EQUIPMENTS
a) Micro well plate
 Flat bottom polystyrene plate, contains 8×12 wells holding 350µL.

b) Multipipette
 An 8 channel 100µL pipette is a good help for even small – scale work.

c) Washing device
 Manually operated washing device.
 May be of use particularly when there is a risk that the samples tested in ELISA
contain infectious materials, so must be collected for subsequent disinfection.

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 d) Micro plate washer
 These are very efficient with unusually low carry-over contamination.

REAGENTS USED

REAGENTS COMPOSITION
Coating buffer 0.01M Phosphate buffer+0.15 M NaCl(PBS)
Diluting / washing buffer 0.01M Phosphate buffer + 0.50M NaCl + 0.1%
tween 20
Blocking buffer Bovine Serum Albumin (BSA)
Enzyme Horse Reddish Peroxidase (HRPO)
Chromogenic substrate Trimethyl benzidine (TMB)
Stop solution 0.5M H2SO4

PROCEDURE
 Add 25µl standards / samples into the respective micro well.
 Add 50µl Enzyme Conjugate in each well.
 Apply plate sealer and incubate for 45 minutes at 18 - 25ºC
 Wash micro well 5 times with 350µl of diluted wash buffer.
 Add 100µl substrate in each well.
 Incubate for 20 minutes at 18-25ºC in dark.
 Add 100µl stop solution in each well.
 Gently mix for 10 seconds until the blue color completely changes to yellow.

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  Read the Optical Density at 450/630 nm with a microtiter plate reader within
15minutes.

TYPES OF ELISA
ON THE BASIS OF DETECTION
1. Colorimetric ELISA
 Assay to determine the antibody concentration.

2.Chemiluminescent ELISA
 Assay for the quantitation of an antigen in a biological sample.

3. Competitive Fluorescence ELISA

 Assay for the concentration of target antigen is determined by detection of signal
interference.

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ON THE BASIS OF PROCEDURE
1. NON – COMPETITIVE
a) Direct ELISA
 It uses a primary labeled antibody that react directly with the antigen.
 It can be performed with the antigen that is directly immobilized on assay plate.
 Not widely used but common for immuno – histochemical staining of cells and
tissues.

b) Indirect ELISA
 It utilizes a primary unlabeled antibody in conjunction with a labeled secondary
antibody.
 Secondary antibody has specificity for primary antibody.

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c) Sandwich ELISA
 Antigens like tumor markers, hormones , serum proteins may be determined.
 Antigens in the sample bind with the capture antibody and become immobilized.
 The antibody of the enzyme conjugate bind with the immobilized antigen to form a
sandwich of Ab – Ag – Ab / enzyme bound to microwell.

2. COMPETITIVE
 Antibody coated microwell.
 Serum antigen and labeled antigen added together.
 Ab –Ag enzyme complex bound is inversely related to the concentration of antigen
present in sample.
 Increased serum antigen results in reduced binding of Ag – enzyme conjugate with
the antibody producing less enzyme activity and color formation (yellow).
 Used to determine small molecules like T3 , T4 and Progesterone.

3.MULTIPLE & PORTABLE

 A newer technique uses an solid phases made up of an immuno – sorbent polystyrene
rod with 8 – 12 protruding ogives pins.

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  Washing and incubation with antigens / antibodies and chromogen are performed by
dipping the ogives pins into pre – filled microwells with reagents.

READING
 Measures the absorbance at 450nm with the help of ELISA reader.
 Calculate the absorbance for each sample and reference.
 Ascent software for the calculation of results can be used.

TROUBLE SHOOTING IN ELISA

If the negative controls are giving positive results:
 Contamination of the substrate solution, enzyme-labeled antibody, controls them.
 Inadequate rinsing of plates.
 Inadequate blocking of plates.
If no color has developed for the positive controls or for the samples
 Check all reagents for dating and storage conditions.
 Micro well plates not coated properly.
 Reagents applied in wrong order or step omitted.
 Enzymes conjugate defective or inhibited by contaminant.
If very little color has developed for positive controls and the test samples
 Check the dilution of the enzyme labeled antibody.
 The concentration of the substrate.
 Wash buffer not adequately drained after every wash step.
 Inadequate incubation times.
 Enzyme conjugate defective or inhibited by contaminant, substrate defective or
contaminated.
 Microwell plates poorly coated.
If color has developed for the test samples but not the positive controls
 Check the source of positive controls, their expiry data and storage.
If the color can be seen, but the absorbance is not high as expected
 Check the wave length.

PRECAUTIONS
 Use of exchange type pipette (always use new tip).

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  Washing.
 Use reservoir for each reagent and label the reservoir.
 Don’t use the same reservoir for multiple reagents.
 Don’t return the reagents to the stock.
 During incubation, well plate should be covered using the plate cover.
 Coating of wells should be proper with the addition of blocking solution.
 Improper coating gives false positive results.

ADVANTAGES
 Reagents are relatively cheap.
 It is highly specific and sensitive.
 No radiation hazards occur during labeling or disposal of waste.
 Easy to perform and quick procedures.
 Equipment is widely available.
 It can be used to a variety of infections.
 It can be used on most type of biological samples like plasma, serum, urine and cell
extracts.

DISADVANTAGES
 Measurement of enzyme activity can be more complex than the measurement of
activity of some type of radioisotopes.
 Enzyme activity may be affected by plasma constituents.
 Kits are not cheap.
 Very specific to particular antigen but won’t recognize other antigens.
 False positive/ negative possible, especially with mutated / altered antigen.

LIMITATIONS
 Results may not be absolute.
 Antibody must be available (poor producer, interference).
 Concentration may be unclear.
 False positive possible (Ab already present).

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APPLICATIONS
Screening donated blood for evidence of viral contamination by
 HIV-1 and HIV-2 (presences of anti-HIV antibodies)
 Hepatitis C (presences of antibodies)
 Hepatitis B (testing for both antibodies and a viral antigen)
Measuring hormone levels
 HCG (test for pregnancy)
 LH (determining the time of ovulation)
 TSH, T3 and T4 (for thyroid function)
Detecting infections
 Sexually – transmitted agents like HIV, syphilis and chlamydia.
 Hepatitis B and C
 Toxoplasma gondii
Detecting illicit drugs.
Detecting allergens in food and house dust.

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 SDS – PAGE

AIM
 To separate the proteins based on their size rather than their charge.

PRINCIPLE
 A method used to separate and analyse macromolecules, such as protein and nucleic
acids. The polyacrylamide gel matrix is a highly porous material that allows for the
separation of proteins based on their size, with smaller proteins migrating more
quickly through the gel than larger proteins. SDS is used as denaturing agent in
PAGE. The (-)ve charged detergent binds to the proteins, which causes them to unfold
and become more elongated, effectively making all proteins the same charge (-)ve .

MATERIALS REQUIRED

Resolving gel acrylamide solution (10ml)

Gel% Water(mL) 30% 1.5M Tris- 10% SDS 10% APS TEMED
Acrylamide HCl, pH (µL) (µL) (µL)
(mL) 8.8(mL)

8% 4.6 2.6 2.6 100 100 10

10% 3.8 3.4 2.6 100 100 10

12% 3.2 4.0 2.6 100 100 10

15% 2.2 5.0 2.6 100 100 10

Stacking gel solution (10ml)

Gel% Water(mL) 30% 1.5M Tris- 10% SDS 10% APS TEMED
Acrylamid HCl, pH (µL) (µL) (µL)
e (mL) 8.8 (mL)

5% 5.86 1.34 2.6 100 100 10

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Reagents required
 Acrylamide/ bis – acrylamide solution
 Gel casting buffers
 2X Laemmli loading buffer
 10X Running buffer
 10% SDS
 10% Ammonium persulphate
 TEMED
Sample preparation
a) Loading or sample buffer
 SDS
 β – mercaptoethanol
 Bromophenol blue
 Glycerol
b) Protein sample
 Heat the protein sample for 5 minutes at 95ºC with loading dye.
c) Vertical electrophoresis chamber
d) Power pack
e) Glass plates
f) Spacers
g) Combs

GEL STAINING
Staining of protein gels with Coomassie Brilliant Blue R – 20 is a common procedure
to visualize proteins resolved by SDS – PAGE. It is highly sensitive and is suitable for long –
term storage of the gels.
Gel Fix Solution (500mL)
 Methanol – 250Ml
 Glacial acetic acid – 50mL
 Water – 200mL
Coomassie solution
 CBB R – 250 – 0.1%
 Methanol – 40%
 Glacial acetic acid – 10%

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  Filter the stain solution using Whatmann No.1 filter paper.
Destain solution
 Methanol – 50mL
 Glacial acetic acid – 35mL
Gel storage solution
 Glacial acetic acid – 25mL
 Water – 475mL

RESOLVING GEL PREPARATION

 Clean the glass plate and spacers of the gel casting unit with deionized water and
ethanol.
 Assemble the plates with the spacers on a stable, even surface.
 Prepare resolving gel solution (10mL) depending on the percentage of gel required.
 Pour the gel solution in the plates assembled with spacers.
 To maintain an even and horizontal resolving gel surface, overlay the surface with
water or isopropanol.
 Allow the gel to set for about 20 – 30 minutes at room temperature.
STACKING GEL PREPARATION
 Prepare stacking gel solution using the following volumes (for 10mL)
 Discard the overlayed water or isopropanol on the resolving gel.
 Insert the comb immediately ensuring no air bubbles are trapped in the gel or near the
wells.
 Allow the gel to set for about 20 – 30 minutes at room temperature.

SAMPLE PREPARATION
 To a volume of protein sample (cell or tissue lysate), add equal volume of loading
buffer.
 Boil the above mixture at 95ºC for 5 minutes.
 These samples can be stored at -20ºC or may be used to proceed with gel
electrophoresis.

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GEL STAINING
 After the electrophoresis, place the gel in a plastic tray containing gel fix solution.
 Place the tray on a rocking table and fix the proteins for 2 hours.
 Place on a rocking table and stain the gel for 2 – 4 hours.
 After the staining step, wash the gel several times with distilled water to remove
excess stain.
 Add destain solution to the gel.
 Place on rocking table and destain for about 4hours till clear blue bands on clear
background are visible.
 After destaining, the gels may be stored in gel storage solution and photographed as
required.

ADVANTAGE
 Stage chemically cross link gel.
 Sharp bond formation.
 Good for separation of low molecular weight fragment.
 DNA recovered for polyacrylamide is extremely pure.

DISADVANTAGES
 Toxic monomers.
 Gels are chance to leak.
 Difficult to prepare &handle.
 Need new gel for each experiment.

APPLICATION
 Detection of protein.
 Measuring molecular weight.
 Peptide mapping.
 Estimation of protein size, structure, & quantitation.
 Used in western blotting.

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 ISOLATION OF RNA

AIM
 To isolate the RNA from the given sample.

PRINCIPLE
 RNA (Ribonucleic acid) is a polymeric substance present in living cells and many
viruses, consisting of a long single-stranded chain of phosphate and ribose units with
the nitrogen bases adenine, guanine, cytosine, and uracil, which are bonded to the
ribose sugar. RNA is used in all the steps of protein synthesis in all living cells and
carries the genetic information for many viruses. The isolation of RNA with high
quality is a crucial step required to perform various molecular biology experiment.
TRIzol Reagent is a ready-to-use reagent used for RNA isolation from cells and
tissues. TRIzol works by maintaining RNA integrity during tissue homogenization,
while at the same time disrupting and breaking down cells and cell components.
Addition of chloroform, after the centrifugation, separates the solution into aqueous
and organic phases. RNA remains only in the aqueous phase. After transferring the
aqueous phase, RNA can be recovered by precipitation with isopropyl alcohol. But
the DNA and proteins can recover by sequential separation after the removal of
aqueous phase. Precipitation with ethanol requires DNA from the interphase, and an
additional precipitation with isopropyl alcohol requires proteins from the organic
phase. Total RNA extracted by TRIzol Reagent is free from the contamination of
protein and DNA.

MATERIALS REQUIRED
Reagents
 Chloroform (without any additives, such as isoamyl alcohol)
 Isopropyl alcohol
 Trizol
 75% Ethanol (in DEPC-treated water)
 RNase-free water or 0.5% SDS solution.

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PROCEDURE
 To 100 mg of fish tissue sample 100 mg add 1mL of trizol reagent.
 Homogenize the mixture and incubate at room temperature for 5 minutes.
 To this mixture, 200µL chloroform is added and shake vigorously for 20-30 sec.
 The solution is incubated at room temperature for 2-3 min and centrifuged at 1200 g
for 15 minutes at 4℃.
 The aqueous layer is separated to the 1.5mL tube (3 layer separation aqueous layer,
white layer and pink layer).
 Add 500 µL of isopropanol to the separated layer, mix well and incubate at room
temperature at 10 minutes.
 The solution is centrifuged at 12000 rpm for 10 minutes at 4℃
 The supernatant is discarded and Add 750 µL of 75% of ethanol and centrifuged at
12000 rpm for 10 minutes at 4℃.
 The supernatant is discarded and air dry the pellet for 10 seconds.
 To the pellet add 40-50 µL DEPC treated water (depends on the pellet) and keep it at
room temperature for 10 minutes.
 It is then stored at -20 ℃ for overnight.
 Purity check is to be done the next day (overnight) – recommended.

RNA QUANTIFICATION AND PURITY CHECK

RNA concentration = A260 × RNA factor × Dilution factor × Volume of sample

1µl of isolate RNA + 49µl of water = 50µl
OD value of sample T = 0.595
OD value of sample E = 0.519
RNA concentration for 2ml of T sample
= 0.595×40×50×0.02
= 23.8µg/ml
RNA concentration for 2ml of E sample
= 0.519×40×50×0.02
= 20.76µg/ml
Convert it into 1ml of T sample:
1000ng÷X = 23800÷1µl
X = 1000 ÷ 23800

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 X = 0.04201ml
Convert it into 1ml of E sample;
1000ng÷X = 20760÷1µl
X = 1000 ÷ 20760
X = 0.0481ml

PCR (POLYMERERASE CHAIN REACTION)

INTRODUCTION
 Polymerase chain reaction (PCR) is a biochemical and molecular biological technique
for enzymatically replicating DNA without a living organism developed by Kary
Mullis in 1983. Like using living organism. The technique always a small amount at
DNA to be amplified exfantially. As PCR is an intro technique it can be performed
without restriction at the DNA and it can be extensively modified to perform wide
many at genetic manipulation.
PRINCIPLE
 The PCR technique is based on the enzymatic replication of DNA. In PCR, a short
segment of DNA is amplified using primer mediated enzymes. DNA Polymerase
synthesis new strands of DNA complementary to the template DNA. The DNA
polymerase can add a nucleotide to the pre-existing 3’-OH group only. Therefore, a
primer is required. Thus, more nucleotides are added to the 3’ prime end of the DNA
polymerase.
DENATURATION
 In this step to the two stands open to from single stranded DNA all enzymatic reaction
gel stopped. It is carried out at 92 -94 ℃.
ANNEALING
 Annealing t primer to reach original strand for new stand synthesis is carried out
between 45 - 60 ℃.
EXTENSION
 The taq polymerase adds d NTPS, complementary to the template at 3 end at primary
at 72 ℃. Single both strands are copied during PCR. There is an exponatiol increase

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 in member at copies at the gene these steps can be repeated for 20-30 min time in and
cool the reaction mixture the polymerization rale fat taq polymerase is 2000
nucleatide. 1µL and its sterilized gel mould com gel electrophoresis apparalves power
unit and, 1x reaction buffer, 10x TE buffer.
MATERIALS REQUIRED
 The key ingredients of a PCR reaction are Taq polymerase, primers, template DNA,
and nucleotides (DNA building blocks). The ingredients are assembled in a tube,
along dartcooling that allow DNA DNA to be synthesized.
REAGENT
 PCR requires five basic reagents, including DNA template, forward and reverse
primers, DNA polymerase, deoxynucleotide triphosphates (dNTPs) and reaction
buffer.
PROCEDURE
 The standard PCR reaction consist of 2:5 mm MgCl2, 1x buffer, 1mm d NTP each
1mole at each primer ,1unit TAQ polymerase 5 µl al DNA template per tube (DNA
concentration should be in 0.1- 2.0 mg/ml ).

PCR PROGRAMME
 Initial denaturation at 95℃ for 5 min followed by ,
 54℃ for 2min (annealing)
 72 ℃for extension repeat cycle for 4 times
 94 ℃ for 2 min
 55℃ for 30 min
 72℃ for 2 min.
 Repeat cycle for 29 times
 72℃ for 10 min (final extension)
 The annealing temperature for primer should be 2 les then lower annealing at both
forward and reverse primer. The normal extension temperature used is 1 min at is
carried 3 hrs.
FACTORS AFFECTING AMPLIFICATION
 Efficient amplification at target sequences depends on individual reaction component
time and temperature condition at the PCR reaction.

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Sample volume
 20, 50µl volumes are recommend for PCR as it assures adequate thermal equilibrium
at the reaction mixture.
Template DNA
 The purity and the concentration at the specificity a PCR typically transform amount
at plasmid and microgram amount at genomic DNA are used for amplification higher
amount at template DNA inhibit PCR and or result in non- specific amplification. A
number at contaminants svchas SDS, phenol and other reagent used in the template
preparation can inhibit PCR.
Primer
 Primer are oligonucleotide ranging. From 15 - 30 base long. It is one at the most
important factor affecting the quality at PCR. Two Primers are generally used at equal
content -ration in the range at 0.1- 1ml Typically H0-65 GC content is recommended
to avoid internal secondary structure in the primer. Optimal annealing temperature is
empirically by calculating Tm that heat should be same for both forward and Reverse
primer.
Deoxynucleotide Triphosphate
 They mayor Source at Phosphate Source in the reaction mix and change in their
concentration affects the concentration at Mg 2+ typically. The final concentration at
each dNTP in a standard amplification reaction mizture is 200µm. It is Important to
keep dNTP concentration above the estimated km at each dNTP and balanced for best
base in corporation.
Master mix
 The master mix contains the tag master. PCR remember enhancer which enhances
yield and robustness against PCR. Inhibitor such as humic acid and blood
compound.Magnesium concentration is one at the critical parameters in a PCR
reaction as Taq polymerase activity. Depends on Mg2+ ion concentration lower
Mg2+. Concentration may result in unwanted products. PCR reaction can be
optimized with the separate 2mm Mg2+ solution provided.

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TAQ POLYMERASE
 Taq Polymerase is a 94 koa thermostable RNA polymerase isolated from a
thermophilic bacterium Thermus aquaticus. The Solubility at Taq DNA Polymerase at
high temperature allows repeated amplification cycles with one time addition at the
enzyme at the start at the reaction Taq Polymerase has 5’and 3' β nuclease activity and
5’ + 3’ polymerase activity for most amplification reaction. 1.5 to 2 units at enzymes
is recommended as higher enzyme concentration leads to non-specific amplification.

SERUM SEPARATION

BACKGROUND
 The fluid and solute component of blood which does not play a role in clotting. It may
be defined as blood plasma without the clotting factors, or as blood with all cells and
clotting factors removed.
 Blood plasma without the fibrinogen proteins.
 Serum includes all proteins that have no role in bloodclotting (coagulation),
electrolytes, antibodies, antigens, hormones, and any extra substances (such as drugs
and microorganisms).

PROCEDURE
 The blood that is collected from the animals during disssection is taken.
 Collected bood is immediately added with EDTA to avoid clotting
 The blood in the centrifuge tube is centrifuged at 3000rpm for 20 mins at 4c.
 The supernatant contains the serum and the pellet is discarded.
 The supernatant is collected in a centrifuge tube seperately for further studies.

RESULT
 The serum was seperated from the blood that is collected from the animals during
disssection.

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HOMOGENISATION
 Homogenization, in cell biology or molecular biology, is a process whereby different
fractions of a biological sample become equal in composition.
 Process used to disrupt cells and tissues by mechanical means, such as grinding,
blending, or shaking.
 It is commonly used in biology and biochemistry research to extract cellular
components, such as proteins and nucleic acids, for further analysis.

CONCLUSION

Molecular techniques help to minimize the false positive results by targeting the specific
molecule of interest. To identify the biofilm composition to genus level and determine shifts
in the community due to environmental changes. It develops and improves drugs, vaccines,
therapies and diagnostic tests that will improve human and animal health. It allows more
efficient typing of all pathogens and permit discrimination between strains of organisms that
were previously phenotypically identical or uncharacterizable.

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 PHASE – II
[ANIMAL HANDLING]

INTRODUCTION

In this phase, we discussed about the animal handling, experiments and its caring.
Physiologically and anatomically there is similarity between the humans and animals at
organs and organ system, which functions in the similar fashion. This similarity makes
animal ideal for the study and development of products and techniques for humans. By using
laboratory animals, various discoveries have been made such as, diptheria and polio vaccine,
insulin for the treatment of diabetes mellitus, heart valve replacement, antibiotic therapy,
manic depressive drugs etc.

ANIMAL EXPERIMENT & ANIMAL CARING

ETHICAL CERTIFICATE
 Designed to give you a solid understanding of ethics and how they are applied in the
business world. The central government has constituted a Committee for the Purpose
of Control and Supervision of Experiments on Animals (CPCSEA) which is duty
bound to take all such measures as may be necessary to ensure that animals are not
subjected to unnecessary pain or suffering before, during or after the performance of
experiment on them.
 Formed in 1998 and amended during 2001 and 2006 to regulate the experimentation
on animals.

GOALS
 To promote the humane care of animals used in biomedical and behavioral research
and testing.
 To provide specification that enhances animals well-being and quality of research.

OBJECTIVE
 Effective functioning of institutional animal ethical committee.

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MAIN ACTIVITIY
 Registration of establishments – for experiments on animals and for breeding of
animals.
 Approval of Animal House Facilities.
 Permission of Committee for Conducting Experiments.

CPCSEA GUIDELINES
Veterinary care
 Provide by a veterinarian.
 Daily observation of animals adopted.
Quarantine, Stabilization and Separation
 Quarantine period for small lab animals: one week to one month and for large lab
animals: up to 6weeks.
 Physiologic, psychological and nutritional stabilization required.
 Physical separation of animals by species to prevent anxiety and behavioral changes.

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EXPERIMENTAL AREA
 Experiments should be carried out in a separate area away from the place where they
are housed.
 Separate functional areas for surgical support, treatment of animals, post-operative
and intensive care.

PHYSICAL FACILITIES FOR ANIMALS

Building materials Durable, moisture – proof, fire resistant and pest resistant

Corridor Wide enough to facilitate the movement of personnel and

equipment

Animal room doors Should fit properly and should not be rust

Floors Smooth, moisture proof, non-absorbent, skid proof floors

Drains Proper drainage, floors should be sloped

Storage areas Designed for feed, bedding, cages and materials not in use

Temperature Between 18 - 29ºC

Humidity control Range of 30 – 70%

Ventilation Designed with 12 – 15 air cycles per hour

Power and lighting System should be safe

Fluorescent lights are efficient

Emergency backup needed

Noise control Noise free environment

Concrete walls are more effective

ANIMAL HUSBANDRY
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Caging or housing system
The housing system should provide
 Adequate space, freedom of movement, normal postural adjustments.
 Comfortable environment.
 Easy access to food and water.
 Adequate ventilation.

Food and water

 Food should be palatable, non – contaminated and nutritionally adequate.
 Should have continuous access to fresh, potable uncontaminated drinking water.

Feed should contain Feed should not contain

 Moisture.  Insecticides, hormones, antibiotics,

 Crude fibre, crude protein and fumigants or potential toxicants.
crude fat.  Heavy metals
 Essential bits, minerals,
carbohydrates

Bedding
 Should be absorbent, free from toxic chemicals.
 Should be removed and replaced periodically with fresh materials.
 Ideal to change the bedding twice a week.
Surveillance
 Observe for the sign of disease injury or abnormal behavior.
 Animals that shows signs of a contagious disease should be isolated from healthy
animals in the colony
HANDLING & HOLDING
 HANDLING: how human work without hurting.
 HOLDING: used to control the animals during the experiments. While holding the
animal stay calm, move slowly & avoid loud noise.

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TYPES OF HOLDING
Scruff holding
 Suitable for small and calm animals.

V holding
 Convenient for vigorous animal.
 Increase more stability.
 Single handed difficult treatment.

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O holding
 To keep calm the restless animals.

ANIMAL GROUPING
a) Control
b) Negative or induced control
c) Induced + treated
 Each group contains 6 animals.
 Groups can be increased according to our studies.

TREATMENT
By three methods
1) Oral
2) Injection
3) Aroma therapy
1) Oral
 The oral route is the most convenient and usually the safest and least expensive. It has
limitations because of the way a drug typically moves through the digestive tract. For
drugs administered orally, absorption may begin in the mouth and stomach.
2) Injection
 The art of giving medications through the use of a needle and syringe.
4 types of Injection
a) Intraperitoneal injection [8 – 12 hours]
 Drugs are injected in peritoneum (abdomen region).

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b) Intramuscular injection [6 – 8 hours]
 Drugs are directly injected in muscles region.

c) Intervenous injection [instant]

 Drugs are directly injected in blood vessals like in rats and mice, drugs are injected in
tail region.

d) Subcutaneous injection [12 – 24 hours]

 Drugs are injected in between the skin and muscle [fat pad]. Absorption of medicine
from this tissue is slower than in an intramuscle injection.

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3) Aroma therapy
 The therapeutic use of natural essential oils to restore or maintain the well-being
animals. It reduces behavioral issues such as anxieties, obsessive behavior and
aggression, and physical condition such as skin problems, arthritis, allergies and
more.
ANIMAL SACRIFICE
1) Cervical dislocation
 By applying pressure to the neck and dislocating the spinal column from the skull or
brain. To quickly separate the spinal cord from the brain so as to provide the animal
with a fast and painless death.

2) Volatile anesthesia
 Combined ketamine and xylazine gives in over dosage to sacrifice the animal.

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DISSECTION

Animal disposing
 Burry
 Burn

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 ZEBRA FISH

INTRODUCTION
 The zebrafish, Danio rerio is a tropical fresh water fish belonging to the family of
cyprinidae.
 It is a popular aquarium fish, frequently sold under the trade name zebra danio.
 It is an important vertebrate model organism in scientific research.

DISTRIBUTION
 The Zebrsfish is native to the streams of the south eastern Himalayan region.
 It arose in Ganges region in Eastern India and is also native to Pakistan, Bangladesh,
Nepal, and Myanmar.
 It commonly inhabits streams, canals, ditches, ponds, and slow moving to stagnant
water bodies, including rice fields.

TAXONOMY

Kingdom Animalia

Phylum Chordata

Class Actinopterygii

Order Cypriniformes

Family Cyprinidae

Genus Danio

Species rerio

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DESCRIPTION
 The zebrafish is named for the five uniform, pigmented, horizontal, blue stripes on the
side of the body, which are reminiscent of a zebra's stripes, and which extend to the
end of the caudal fin.
 Its shape is fusiform and laterally compressed, with its mouth directed upwards.The
male is torpedo-shaped, with gold stripes between the blue stripes; the female has a
larger, whitish belly and silver stripes instead of gold.
 Adult females exhibit a small genital papilla in front of the anal fin origin.The
zebrafish can reach up to 4–5 cm (1.6–2.0 in) in length, although they typically are
1.8–3.7 cm (0.7–1.5 in) in the wild with some variations depending on location.
 Its lifespan in captivity is around two to three years, although in ideal conditions, this
may be extended to over five years. In the wild it is typically an annual species.

CHALLENGES
1. System maintenance
 Zebrafish are kept in a circulating system that continuously filters and aerates the
system water to maintain the water quality required for a healthy aquatic environment.
The circulating system also helps to filter excess food and fish excreta.
2. Feeding
 Zebrafish can be fed with dry food (food size from 100 microns for larvae to 300/400
microns for adult fish) or live food (brine shrimps). Brine shrimp (Artemia sp.) eggs
are available from local pet shops and can be hatched in the laboratory.
3. Breeding
 The fish are photoperiodic and breed at dawn. Keep the water temperature between 23
and 28 degrees C and a pH between 6.2 to 7.5. For successful breeding: In the late
afternoon of the day before eggs are wanted, cover the bottom of the tank with a
single layer of marbles. zebrafish were placed in breeding tanks in one of three sex
ratios (1 male:1 female; 3 males:1 female; 1 male:3 females).

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4. Raising of Larvae

ADVANTAGES
 Short reproductive cycle.
 High number of offspring.
 High throughput drug screening.
 Zebrafish genome is fully sequenced.
 Over 70% of annotated human genes have a true orthologue in the zebrafish genome

CONCLUSION

The proper care and use of laboratory animals in research, testing, teaching, and
production (animal use) require scientific and professional judgment based on the animals’
needs and their intended use. An animal care and use program (hereafter referred to as the
Program) comprises all activities conducted by and at an institution that have a direct impact
on the well-being of animals, including animal and veterinary care, policies and procedures,
personnel and program management and oversight, occupational health and safety,
institutional animal care and use committee (IACUC) functions, and animal facility design
and management.

BIBLIOGRAPHY
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1) www.ncbi.nlm.nih.gov
2) www.sciencedirect.com
3) www.slideshare.net
4) https://pubmed.ncbi.nlm.nih.gov
5) https://en.m.wikipedia.org
6) https://www.nature.com

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