Training report at Chandigarh University

UNIVERSITY INSTITUTE OF ENGINERRING
DEPARTEMENT OF BIOTECHNOLOGY
INSTITUTIONAL SUMMER TRAINING
DESIGNED BY: ISHANT
GUPTA
Summer Institutional Training-
Basic tools and techniques in biotechnology
Code-21BBI211
Starting date- 20th
June Ending date-15th
July
NAME OF STUDENT: ISHANT GUPTA
UID OF STUDENT: 21BBE101172
DEPARTMENT: DEPARTMENT OF BIOTECHNOLOGY AND FOOD
TECHNOLOGY

DESIGNED BY: ISHANT
GUPTA
CERTIFICATE

DESIGNED BY: ISHANT
GUPTA
ACKNOWLEDGEMENT
It is my immense pleasure to express my sincere gratitude to our esteemed mentor
and supervisor Dr. Navjeevan Dadwal, Assistant Professor, Dr. Mitun
Chakraborty, Professor, Dr. Md. Azizur Rehman, Professor, Dr. Swatantar Kumar,
Assistant Professor, Dr. Deepak Kala, Professor, Dr. Swayamprava Dalai,
Department of Biotechnology Engineering, Chandigarh University, Gharuan
whose guidance, encouragement, suggestions, cooperation and patience throughout
the course of work, helped me in the accomplishment of this project.
I offer my humble thanks and shall always be obliged to him. I am grateful
to Professor Dr. Vinay Dwivedi, Head of the Department of Biotechnology
Engineering and Food Technology, Chandigarh University, Gharuan for timely
providing all the facilities.
I would also like to extend my thanks to the technicians of the laboratory of
“Biotechnology Department” for offering me the resources for the running
program.
I wish to thanks my parents for their support and encouragement throughout my
study.

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GUPTA
CONTENTS
PART-1 ( MOLECULAR BIOLOGY )
1.1 DNA Isolation ..……………………………………………………………………….… 5-8
1.2 Gel Electrophoresis ...……………………………………………………………….….. 9-17
1.3 Spectrophotometry .……………………………………………………………............ 18-24
1.4 Polymerase Chain Reaction…………………………………………………….……... 25-27
PART-2 ( INSTRUMENTATION )
2.1 Hot Air Oven, Autoclave, Gel Doc, PCR, Laminar Air Flow, Microscope, Centrifugator,
Incubator, Spectrophotometer ...………………………………………………………... 28-37
PART-3 ( MICRO BIOLOGY )
3.1 Spreading ...…………………………………………...………………………………. 38-42
3.2 Streaking ……………………………………………………………………...………. 43-44
3.3 BSL ……………………………………………………………………..…………….. 45-47
3.4 Ocular Microscopy …………………………………………………………...………. 48-49
PART-4 ( BIO CHEMISTRY )
4.1 Chromatography ………….…………………………………………………………... 50-52
4.2 Chlorophyll Extraction ..……………………………………………………………... 53-55
PART-5 ( BIOINFORMATICS )
5.1 Database Searching from NCBI for Data Retrieval FASTA Analysis ...……………... 56-63
5.2 Database Searching from NCBI for Data Retrieval BLAST Analysis ...……………... 64-72
5.3 Retrieving Data of Five Same Species for Multiple Genome Sequencing to Get their
Phylogenetic Relationship ...……………………………………………………….…….73-82
5.4 To Retrieve a Protein Sequence from POB Database and Analyze its 3D Structure … 83-94

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PART-1 ( MOLECULAR BIOLOGY )
1.1DNA Isolation
 AIM:
To extract genomic DNA from human blood.
 MATERIAL REQUIRED:
Name Quantity
Solution A 1ml
Solution B 1ml
Solution C 1ml
Micro centrifuge 1
UV trans illuminator 1
Water bath 1
Distilled Water
Micropipette 1

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 CHEMICALS USED:
Tris HCl, Potassium Chloride, Magnesium Chloride, EDTA, Sodium Chloride, Sodium dodecyl
sulphate(SDS), Isopropanol, Ethanol
 THEORY:
Blood is a complex assemblage of cells, proteins, metabolites, and other substances. Erythrocytes
make up more than 99 percent of the cells in human blood, or around 56 percent of its volume.
Because they lack nuclei, human erythrocytes and thrombocyte platelets are not appropriate for
DNA production. The only blood cells having nuclei are leukocytes and white blood cells
(WBC), and their presence in various blood samples depends on the donor's health.
The EDTA buffer, which is used as an RBC lysis buffer, comprises salts and detergents that
create a hypertonic environment that lyses RBCs.
To lyse WBC, a buffer comprising detergents that effectively remove the contained protein and
guanidium thiocynate salt is utilized.

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 PROCEDURE:
1. Use 10 ml blood sample containing 0.5 ml of 0.5 molar EDTA as anticoagulant stored at 4°C
2. Centrifuge at 3000 R.P.M. for 20 minutes and remove the supernatant.
3. Mix the pallet with 3 volume of RBC lysis buffer (17 millimolar Tris HCl, pH 7.65 and 140
millimolar ammonium chloride) and keep for 15 min at 37°C..
4. Centrifuge at 2000 R.P.M. for 20 minutes at room temperature. Discard the supernatant
5. Pallet is resuspended in sterile normal saline and centrifuge at 2000 R.P.M. for 10 minutes.
6. Pallet is suspended in DNA extraction buffer (3 ml/10 ml blood) using vortex mixer.
7. Add 10% SDS (200 microliter per 10 ml) and mix carefully by inverting the tube several
times.
8. 8. Proteinase K (20 milligram per ml in distilled water) is mixed at 30 μl per 10 ml blood in
2
9. Equal volume after 1" addition mixture is incubated at 50°C for 2 hours and after 2nd
addition mixture is incubated at 50°C overnight.
10. Equal volume of Tris saturated phenol pH 7.8 is added and mixed by inverting the tubes
gently for 15 minutes then the mixture is centrifuged at 5000 rpm for 30 minutes at room
temperature.
11. The upper aqueous phase is taken out and 3 ml of phenol chloroform Iso amyl alcohol
(25:24:1) and centrifuge at 5000 rpm for 30 minutes
12. Upper aq. Phase is transferred to another centrifuge tube and the DNA is precipitated by the
3 volume of absolute alcohol.
13. Precipitated DNA is spooled out with the help of micropipette tip
14. Spooled DNA is transferred to 70% alcohol and centrifuged at 12000 rpm for 10 minutes and
supernatant is discarded.
15. Now DNA pallet is dissolved in 200 micro liter TE buffer (pH-8) in micropipette.

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 OBSERVATION:
Successful isolation of the blood sample's DNA was accomplished. At the vial's bottom, DNA
strands could be seen. After centrifugation, the pellet was composed of DNA strands.
 INFERENCE:
The current method is suitable for DNA extraction from both human and animal blood samples
in any molecular biology labs due to its high quality and quantity, lack of enzymatic processing,
and consequently low cost.
 PRECAUTIONS:
1. Well measured amount of solutions should be added.
2. DNA handling was done very gently to avoid mixing.
3. RBC lysis was repeated multiple times.
4. Perform Extractions at 4°C, on ice or in the cold.
5. Store purified DNA correctly.

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1.2 Gel Electrophoresis
 AIM:
To analyze the given nucleic acid using Agarose Gel Electrophoresis.
 APPRATUS:
1. Agarose solution
2. Ethidium bromide
3. Electrophoresis buffer
4. 6x gel buffer
5. DNA sample
6. DNA size standard.
 PRINCIPLE:
The separation medium is a gel made from agarose. Agarose is isolated from the seaweed genera
Gelidium and Gracilaria and consists of repeated agarobiose (L- and D-galactose) subunits.
During gelation, agarose polymers associate non-covalently and form a network of bundles
whose pore sizes determine a gel’s molecular sieving properties. In general, the higher the
concentration of agarose, the smaller the pore size.

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 PROCEDURE:
 Preparation of the Gel
1. Weigh out the appropriate mass of agarose into an Erlenmeyer flask. Agarose gels are
prepared using a w/v percentage solution. The concentration of agarose in a gel will depend
on the sizes of the DNA fragments to be separated, with most gels ranging between 0.5%-
2%. The volume of the buffer should not be greater than 1/3 of the capacity of the flask.
2. Add running buffer to the agarose-containing flask. Swirl to mix. The most common gel
running buffers are TAE (40 mM Tris-acetate, 1 mM EDTA) and TBE (45 mM Tris-borate,
1 mM EDTA).
3. Melt the agarose/buffer mixture. This is most commonly done by heating in a microwave, but
can also be done over a Bunsen flame. At 30 s intervals, remove the flask and swirl the
contents to mix well. Repeat until the agarose has completely dissolved.
4. Add ethidium bromide (EtBr) to a concentration of 0.5 μg/ml. Alternatively, the gel may also
be stained after electrophoresis in running buffer containing 0.5 μg/ml EtBr for 15-30 min,
followed by destaining in running buffer for an equal length of time.
 2. Setting up of Gel Apparatus and Separation of DNA Fragments
1. Add loading dye to the DNA samples to be separated (Fig. 2). Gel loading dye is typically
made at 6X concentration (0.25% bromphenol blue, 0.25% xylene cyanol, 30% glycerol).
Loading dye helps to track how far your DNA sample has traveled, and also allows the
sample to sink into the gel.

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2. Program the power supply to desired voltage (1-5V/cm between electrodes).
3. Add enough running buffer to cover the surface of the gel. It is important to use the same
running buffer as the one used to prepare the gel.
4. Attach the leads of the gel box to the power supply. Turn on the power supply and verify that
both gel box and power supply are working.
5. Remove the lid. Slowly and carefully load the DNA sample(s) into the gel (Fig. 3). An
appropriate DNA size marker should always be loaded along with experimental samples.
6. Replace the lid to the gel box. The cathode (black leads) should be closer the wells than the
anode (red leads). Double check that the electrodes are plugged into the correct slots in the
power supply.
7. Turn on the power. Run the gel until the dye has migrated to an appropriate distance.
 3. Observing Separated DNA fragments
1. When electrophoresis has completed, turn off the power supply and remove the lid of the gel
box.
2. Remove gel from the gel box. Drain off excess buffer from the surface of the gel. Place the
gel tray on paper towels to absorb any extra running buffer.
3. Remove the gel from the gel tray and expose the gel to uv light. This is most commonly done
using a gel documentation system (Fig. 4). DNA bands should show up as orange fluorescent
bands. Take a picture of the gel (Fig. 5).
4. Properly dispose of the gel and running buffer per institution regulations.
 Representative Results
Figure 5 represents a typical result after agarose gel electrophoresis of PCR products. After
separation, the resulting DNA fragments are visible as clearly defined bands. The DNA standard
or ladder should be separated to a degree that allows for the useful determination of the sizes of

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sample bands. In the example shown, DNA fragments of 765 bp, 880 bp and 1022 bp are
separated on a 1.5% agarose gel along with a 2-log DNA ladder.
.
Figure 1. A solidified agarose gel after removal of the comb.

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Figure 2. A student adding loading dye to her DNA samples.

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Figure 3. A student loading the DNA sample into a well in the gel.
Figure 4. An example of a gel documentation system.

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Figure 5. An image of a gel post electrophoresis. EtBr was added to the gel before
electrophoresis to a final concentration of 0.5 μg/ml, followed by separation at 100 V for 1 hour.
The gel was exposed to uv light and the picture taken with a gel documentation system.
 DISCUSSIONS:
The exact sizes of separated DNA fragments can be determined by plotting the log of the
molecular weight for the different bands of a DNA standard against the distance traveled by each
band. The DNA standard contains a mixture of DNA fragments of pre-determined sizes that can
be compared against the unknown DNA samples. It is important to note that different forms of
DNA move through the gel at different rates. Supercoiled plasmid DNA, because of its compact
conformation, moves through the gel fastest, followed by a linear DNA fragment of the same
size, with the open circular form traveling the slowest.

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 INFERENCE:
In inference or conclusion, since the adoption of agarose gels in the 1970s for the separation of
DNA, it has proven to be one of the most useful and versatile techniques in biological sciences
research.
 PRECAUTIONS:
1. When locating, or working around or near an electrophoresis unit, avoid unintentional
grounding points and conductors (e.g., sinks and other water sources, metal plates, aluminum
foil, jewelry, pipes, or other metal equipment).
2. Always think and look before touching any part of the apparatus.

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1.3 Spectrophotometry
 AIM:
By using spectrophotometry determine the concentrations of red and blue dyes as standards.
 APPARATUS/INSTUMENTS REQUIRED:
Vernier spectrophotometer , Distilled water, Salt, Beaker, Cuvette tissue for cleaning cuvette
surfaces, Cuvettes for spectrophotometer, Petri dish or clear plastic cup, Pipette ,Vernier light
sensors
 BASIC CONCEPT:
Spectrophotometer is often used to study solutions. A solution containing an absorbing material
is compared to a reference solution of the same solvent and non-absorbing materials. The
transmittance of the reference solution is set to 100% (Abs = 0), then the relative transmittance
of the solution is measured.
 THEORY:
Every chemical compound absorbs, transmits, or reflects light (electromagnetic radiation) over a
certain range of wavelength. Spectrophotometry is a measurement of how much a chemical
substance absorbs or transmits. Spectrophotometry is widely used for quantitative analysis in
various areas (e.g., chemistry, physics, biology, biochemistry, material and chemical

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engineering, clinical applications, industrial applications, etc). Any application that deals with
chemical substances or materials can use this technique. In biochemistry, for example, it is used
to determine enzyme-catalyzed reactions. In clinical applications, it is used to examine blood or
tissues for clinical diagnosis. There are also several variations of the spectrophotometry such as
atomic absorption spectrophotometry and atomic emission spectrophotometry.
A spectrophotometer is an instrument that measures the amount of photons (the intensity of light)
absorbed after it passes through sample solution. With the spectrophotometer, the amount of a
known chemical substance (concentrations) can also be determined by measuring the intensity of
light detected. Depending on the range of wavelength of light source, it can be classified into two
different types:
• UV-visible spectrophotometer: uses light over the ultraviolet range (185 - 400 nm) and
visible range (400 - 700 nm) of electromagnetic radiation spectrum.
• IR spectrophotometer: uses light over the infrared range (700 - 15000 nm) of
electromagnetic radiation spectrum.
In visible spectrophotometry, the absorption or the transmission of a certain substance can be
determined by the observed color. For instance, a solution sample that absorbs light over all
visible ranges (i.e., transmits none of visible wavelengths) appears black in theory. On the other
hand, if all visible wavelengths are transmitted (i.e., absorbs nothing), the solution sample
appears white. If a solution sample absorbs red light (~700 nm), it appears green because green is
the complementary color of red. Visible spectrophotometers, in practice, use a prism to narrow
down a certain range of wavelength (to filter out other wavelengths) so that the particular beam
of light is passed through a solution sample.
A spectrophotometer, in general, consists of two devices; a spectrometer and a photometer. A
spectrometer is a device that produces, typically disperses and measures light. A photometer
indicates the photoelectric detector that measures the intensity of light.

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Spectrometer: It produces a desired range of wavelength of light. First a collimator (lens)
transmits a straight beam of light (photons) that passes through a monochromator (prism) to split
it into several component wavelengths (spectrum). Then a wavelength selector (slit) transmits
only the desired wavelengths.
Photometer: After the desired range of wavelength of light passes through the solution of a
sample in cuvette, the photometer detects the amount of photons that is absorbed and then sends
a signal to a galvanometer or a digital display.
 PROCEDURE:
1. 1.Start the program and enter your assigned code #.
2. Turn on the instrument by clicking on the knob at the left front. Manipulate this knob
to set the Transmittance to 0.0.
3. Prepare a standard solution of RED dye by clicking on the arrow beside the red rectangle
at the top of the page, and then click the mouse on the squirt bottle to put a solution of red
dye in Cell #1. Record the concentration of this red dye solution (ppm).
4. Prepare a solution of BLUE dye in Cell #2. Record the concentration of this blue dye
solution.
5. Click on Cell #1 to place the red dye standard in the cell compartment.
6. Click on SCAN. The observed data will pop up in a gray window. Close that window.
7. Click on the cell compartment or on one of the cells to return Cell #1 to the rack.
8. Click on Cell #2 to place the blue dye standard in the cell compartment.
9. Click on SCAN. The observed data for Cell #1 and Cell #2 will appear.
10. Inspect the data to select a wavelength (WL1) at which the absorbance of the red dye is
strong and that of the blue dye is negligible, then a wavelength (WL2) at which the
absorbance of the blue dye is strong and that of the red dye is negligible.

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11. Set the wavelength to the value you have chosen for WL1. Click on Cell #0 (distilled
water) to place it in the cell compartment. Operate the knob on the right front to set the
absorbance of this solution to 0.000.
12. Place each of the cells in the cell compartment and record their absorbance values at
WL1.
13. Re-set the wavelength to the value you have chosen for WL2. Place Cell #0 in the cell
compartment. Set the absorbance of this solution to 0.000.
14. Place each of the cells in the cell compartment and record their absorbance values at
WL2.
15. Calculate the concentrations of red and blue dye in the unknown solutions.
16. Click on "check results" and see how you've done. If there are errors, re-check your
calculations.
 SIMULATOR LINK:
https://web.mst.edu/~gbert/Color_Lg/color.html?455
 OBSERVATIONS:
RED DYE:

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BLUE DYE:

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SIMULATION:
 RESULT AND SUMMARY:
The concentrations of the solutions of dyes are:
 INFERENCE:

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In this experiment, concentration of blue dye in sample unknown 4 (U4) is more while in sample
unknown5 (U5) concentrations are nearly equal.
 PRECAUTIONS:
1. Take out the desiccant in the sample chamber before starting the machine.
2. The solution in the cuvette should be 2/3～4/5 of the height of the cuvette, and it
should not be too full to prevent the liquid from overflowing and corroding the
instrument.
3. Keep the cuvette clean during the measurement, and wipe the liquid droplets on the
wall with lens cleaning paper.
4. Do not pinch the translucent surface with your hands. When measuring the
ultraviolet wavelength, a quartz cuvette is required.
5. During the measurement, it is forbidden to put reagents or liquid substances on the
surface of the instrument. If the UV-Vis spectrophotometer has solution overflow or
other reasons, the sample tank should be cleaned up as soon as possible.
6. After the experiment, pour out the solution in the cuvette, then rinse the cuvette with
distilled water or organic solvent until it is clean, and dry it upside down. Turn off
the power, put the desiccant into the sample chamber, cover the dust cover, and
register for use.

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1.4 Polymerase Chain Reaction (PCR)
 AIM:
This experiment can be used for exponential amplification of a DNA of interest. There are
different existing variations and applications of the reaction which can be used for special
functions (i.e. addition of certain short sequences at 3 or 5 point end, insertion of point mutation
etc.)
 MATERIALS/INSTRUMENTS REQUIRED:
Q5-High-Fidelity 2x Master Mix (NEB, USA) , Forward primer (See list of sequences), Reverse
primer, template DNA.
 PRINCIPLE OF PCR:
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 synthesises new strands of
DNA complementary to the template DNA. The DNA polymerase can add a nucleotide to the
pre-existing 3’- OH group only.
 THEORY:
PCR is a very powerful amplification tool so very little DNA is actually required (usually in the
pg range). To copy DNA, all polymerases require a short sequence of nucleotides to provide a
free 3’OH group. Within cells of most organisms enzymes unwind the duplex and then RNA
polymerase adds priming nucleotides. This priming allows the attachment of DNA polymerase

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from where extension can occur. In PCR however, there is a different mechanism of replication.
PCR requires that the flanking sequences of the target DNA be known, so that single stranded
synthetic oligonucleotide primers can be generated. Once these have annealed to the target DNA
the polymerase can synthesize the rest of the chain. Primers can be specific to a particular
sequence, or they can be universal to sequences that are very common within a DNA molecule
allowing for a wide variety of DNA templates. Deoxyadenylate (A), deoxythymidylate (T),
deoxyguanylate (G) and deoxyctidylate (C) are components of the reaction mixture that are the
basic building blocks of DNA required for the synthesis of the primers and for their extension.
Note that both the primers and the deoxynucleotides are added to the mixture in excess. The
most commonly used polymerase in PCR is referred to asTaq-polymerase. This enzyme was
originally found in the 1970's from the thermophilic bacterium Thermus aquaticus. Before its
discovery, PCR was a much longer and costlier process since the thermosensitive E.coli DNA
polymerase used lost its activity during the heating process and fresh enzyme had to be added at
every cycle. Owing that T. aquaticus lives in a hot environment (approx. 75°C), the enzymes that
power its internal metabolism have adapted to these conditions resulting in heat-stability. This
polymerase will therefore, retains its activity when the mixture is heated and can be used for
cycle after cycle.
 PROCEDURE:
 INFERENCE:
PCR is important because it can generate several copies of a DNA sequence in a very short time
period. It is also important in forensic science as a tool for genetic engineering. It helps in
analyzing the gene expression. PCR is so sensitive that the DNA present in an individual cell can
be isolated and amplified. This process is faster and less tedious than the traditional methods of
gene cloning.

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 PRECAUTIONS:
1. Do not use any tube or plate that is not appropriate for the PCR machine you are using.
2. Make sure tubes and especially plates are well sealed before you begin run.
3. Clean up any spilled solutions and dispose of in appropriate biohazard boxes.
4. Be careful with PCR machine lids. These can be damaged if you slam or drop lids.
5. Turn PCR machine off when you are done using.
6. Make sure PCR heater block is clean before you start a run. Check each tube receptacle
before you start.
7. Distribute tubes evenly across block so lid will seat flat against top of tubes for even
heating and sealing.

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PART-2 ( INSTRUMENTATION )
2.1 Hot Air Oven, Autoclave, Gel Doc, PCR, Laminar Air Flow,
Microscope, Centrifugator, Incubator, Spectrophotometer
 AIM:
Information of instruments used while performing the experiments.
 MATERIALS/INSTRUMENTS REQUIRED:
Hot Air Oven, Autoclave, Gel Doc, PCR, Laminar Air Flow, Microscope, Centrifugator,
Incubator, Spectrophotometer.
 THEORY/PRINCIPLE:
HOT AIR OVEN:
Electric appliances called hot air ovens use dry heat to disinfect. Louis Pasteur was the person
who first created them. Typically, a thermostat is used to regulate the temperature. Because their
twin walls are metallic on the outside and weak conductors on the inside, they insulate well and
save energy. To help with insulation, there is a space between that is filled with air. The voltage

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and frequency (Hz) being used determine power. Unlike an autoclave, they don't need water, and
the pressure within the oven doesn't build up as high, making them safer to use. They are also
better suited for use in a laboratory setting because of this. Despite being much smaller than
autoclaves, they can be just as efficient.
Precautions:
Make sure you should not use any plastic material to avoid melting.
AUTOCLAVE:

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A device known as an autoclave uses the moist heat sterilization (MHS) method to kill bacteria,
viruses, and even heat-resistant endospores on a variety of instruments by creating saturated
steam under pressure. To do this, the device's components are heated to temperatures higher than
the boiling point of water. Gas laws, which essentially state that the higher the pressure inside the
device, the higher the temperature climbs, are likewise embodied in this process. Because the
moisture in the steam helps coagulate the proteins that germs feed on, steam sterilization is
efficient. The proteins are coagulated, rendering the microorganisms inoperable and ultimately
killing them. Autoclaves normally produce a temperature of about 121 degrees Celsius and finish
the sterilizing process in about 15-20 minutes.
Precautions- Be sure arms are covered by a lab coat and longer heat-resistant gloves to prevent
burns from heat and steam.

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GEL DOC:
A gel doc, often called a gel documentation system, is a piece of equipment frequently seen in
molecular biology labs for photographing and documenting nucleic acids and proteins floating
on polyacrylamide or agarose gels. Ethidium bromide or other nucleic acid stains, such as Gel
Green, are frequently used to stain these gels. An ultraviolet (UV) light trans illuminator, a hood
or a darkroom to block out external light sources and screen the operator from UV exposure, and
a CCD camera for image capture are often included in a gel doc. Low light capable cameras
comprised of CMOS camera sensors, including Sony's Pregius and Exmor series, are also being
used in gel documentation systems thanks to advancements in CMOS camera sensors.
Precautions:
Make sure you have shut down UV Trans illuminator or other lighting in gel imager.

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PCR:
Thermal cyclers, often known as PCR machines, use cyclical routines to control temperature to
amplify DNA. These extremely specialized instruments, which are essential for molecular
applications, have undergone significant design and mechanical improvements over the years,
including improvements to lid security, heat block dependability, hardware functioning, and
other areas. To accept several sample forms, including single tubes, strip wells, and multi-well
tubes, blocks become easily interchangeable. When choosing a thermocycler, the temperature
range is a crucial factor to take into account, with 4°C-99°C being the most typical range. When
considering sample retention time, more sophisticated instruments have a capability that allows
them to cool down to as low as 0°C.
Precautions:
Clean up any spilled solutions and dispose of in appropriate biohazard boxes.

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LAMINAR AIR FLOW:
Equipment with a laminar air flow is frequently utilized in microbiology labs. It comprises of a
chamber with an air blower attached to its back side that allows air to flow in straight, parallel
lines at a constant pace. A laminar flow cabinet/primary hood's function is to create a clean
working environment. It filters and collects all forms of impurity particles entering the cabinet
for this purpose. It utilizes a filter pad and a specialized filter system called a high-efficiency
particulate air filter, or HEPA filter, which can filter out airborne impurity particles as small as
0.3 micrometers.
Precautions:
The laminar flow cabinet should be sterilized with the UV light before and after the operation.

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MICROSCOPE:
A compound microscope may be recognized almost anywhere thanks to its illustrative curved
arm, substantial flat base, and delicate stack of lens-bearing tubes. A microscope primarily
"magnifies" the view of whatever is in its field of focus. When a specimen is placed beneath the
"objective" lens, two lenses in the device work in tandem to enlarge the image of the specimen.
Precautions:
Be aware if your microscope has a mercury lamp.
CENTRIFUGATOR:

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On the basis of centrifugation, the centrifuge operates. Centrifugation is the angular motion-
based process of sedimenting the particles (materials) inside the container (test tube, bucket,
etc.). As a result, it aids in the separation of particles of various sizes and shapes. The particle
will experience a centripetal force toward the center of rotation when it is travelling with a
specific angular velocity. Different particles may sediment at varying speeds. The particle will
arrive at the base more quickly if the sedimentation rate is higher. As a result, this aids in particle
separation.
Precautions:
Lubricate O-rings and rotor threads weekly.
INCUBATOR:

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Every incubator operates under the fundamental tenet that microorganisms need the ideal
environment to live and grow. The ideal temperature, humidity, oxygen, and carbon dioxide are
all present in an incubator, allowing the microorganisms to grow and increase in population. An
incubator's thermostat controls the temperature within the incubator. We can use the
thermometer to check this temperature outside. We keep the temperature within the incubator
constant by using the heating and no-heating cycles.
Precautions:
Avoid humid, damp areas that may be harboring fungal growth.

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SPECTOPHOTOMETER:
A spectrometer and a photometer are the components of a spectrophotometer. The photometer
measures light intensity, while the spectrometer generates light of any wavelength. The liquid or
sample is positioned between the spectrometer and the photometer because of how the
spectrophotometer is constructed. The photometer generates a voltage signal for the display and
calculates the amount of light that enters the sample. The voltage signal also fluctuates when
light absorption varies.
Precautions:
Use only dried air or nitrogen to purge your spectrometer. Never use a flammable, combustible,
or toxic gas to purge this instrument.

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PART-3 ( MICRO BIOLOGY )
3.1 Spreading
 AIM:
To perform spreading technique for culture media.
 APPARATUS:
1. Glassware’s
i. Screw-capped test tubes
ii. Sterile pipettes
iii. Sterile bent glass rods (bent in the shape of a hockey stick), or commercially
available sterile spreaders
2. Medium: Plate count agar or nutrient agar. The surface of the plate must not be too moist because
the added liquid must soak in so the cells remain stationary.
3. Alcohol (ethanol)
 PRINCIPLE:

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A liquid sample is diluted in a series of tubes containing sterile water or physiological saline. A
fixed volume of sample usually 0-1 mL is removed from each tube and placed onto the agar
surface. The sample is then spread evenly over the agar surface by means of a sterile, bent glass
rod.
Following incubation, the dispersed cells develop into isolated colonies. The number of colonies
present on the plate is counted and is multiplied by the dilution factor to calculate the total
number of viable organisms originally present in the sample.
In this method, the substance to be tested if not in liquid form is ground and dissolved in a
suitable liquid medium. The sample is then diluted in 10-fold serial dilutions and plated in an
appropriate medium.
Procedure for Spread Plate Technique
A: Serial dilution
1. Prepare a series of at least 6 test tubes containing 9 ml of sterile distilled water.
2. Using a sterile pipette, add 1ml of sample to the first tube of the set. Label it as 10-1.
3. Mix the contents well by swirling the tube upside down a few times.
4. From the first tube, take 1ml of the sample and transfer it to the second tube. Label it as 10-2.
5. Repeat the procedure with all the remaining tubes labeling them until 10-6
.
B. Plating
1. Pipette out 0.1 ml* from the appropriate desired dilution series onto the center of the surface
of an agar plate. The surface of the agar must not be too moist because the added liquid must
soak in so the cells remain stationary.
2. Dip the L-shaped glass spreader (hockey stick) into ethanol. Ethanol is used to sterilize the
glass spreader.
3. Flame the glass spreader over a bunsen burner.

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4. Spread the sample evenly over the surface of agar using a cool alcohol-flamed glass rod
spreader, carefully rotating the Petri dish underneath at an angle of 45o
at the same time.
5. Incubate the plate at 37°C for 24-48 hours.
 Calculation:
If your spread plate is successful, after incubation you will get isolated countable colonies evenly
spread across the surface of the agar. Count the number of colonies and multiply it by the
appropriate dilution factor to determine the colony-forming units (CFU) present per ml in the
original sample.
CFU/ml = (number of colonies x dilution factor) / volume of culture plated
Serial dilution and number of colonies formed (Image source Biorender.com)
For example, suppose the plate of the 10-4
dilution yielded a count of 27 colonies. Then, the
number of bacteria in 1 ml of the original sample can be calculated as follows:
Bacteria/ml = (27) x (104
) x 10 = 2.7 × 106
(we have multiplied by 10 because we have used 0.1mL while plating the agar plate)

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B
 BENEFITS COMPARE TO POUR PLATE TECHNIQUE:
1. Only surface colonies develop
2. The organisms are not required to withstand the temperature of liquid agar.
 USES:
1. Unlike the streak-plate technique, the spread plate technique can be performed in a
quantitative manner to determine the number of bacteria present in a sample. Spread plate

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technique is most commonly applied for microbial testing of foods or any other samples or to
isolate and identify a variety of microbial flora present in the environmental samples.
 SAFETY PRECAUTIONS:
1. Treat all microorganisms as potential pathogens. ...
2. Sterilize equipment and materials. ...
3. Disinfect work areas before and after use. ...
4. Wash your hands. ...
5. Never pipette by mouth. ...
6. Do not eat or drink in the lab, nor store food in areas where microorganisms are stored
 Limitations of Spread Plate Technique:
1. Strick aerobes are favored while microaerophilic tends to glow slower.
2. Crowding of the colonies makes the enumeration difficult.

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3.2 Streaking
AIM:
How to streak the culture media
THEORY:
Streak plate technique is used for the isolation into a pure culture of the organisms (mostly
bacteria), from a mixed population. The inoculum is streaked over the agar surface in such a way
that it “thins out” the bacteria. Some individual bacterial cells are separated and well-spaced
from each other.
As the original sample is diluted by streaking it over successive quadrants, the number of
organisms decreases. Usually, by the third or fourth quadrant, only a few organisms are
transferred which will give discrete colony forming units (CFUs).
PRINCIPLE:
The inoculum is diluted by streaking it across the surface of the agar plate. While streaking in
successive areas of the plate, the inoculum is diluted to the point where there is only one
bacterial cell deposited every few millimeters on the surface of the agar plate. When these lone
bacterial cells divide and give rise to thousands and thousands of new bacterial cells, an isolated
colony is formed. Pure cultures can be obtained by picking well-isolated colonies and restreaking
these on fresh agar plates. A common assumption is an isolated colony of bacteria is the progeny
of a single bacterial cell (i.e. colony is the clone). However, this is not necessarily true. With

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species in which the cells form a characteristic grouping during cell divisions, the colony-
forming unit may develop from a group of cells rather than form a single cell. For example,
clusters of staphylococci.

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3.3 BSL
 AIM:
To learn the biosafety rules for the lab.
 THEORY:
Biosafety Level:
To isolate hazardous biological agents in a lab setting, a set of biocontainment procedures known
as a biosafety level (BSL), often referred to as a pathogen/protection level, is required. There are
many levels of containment, ranging from the lowest biosafety level 1 (BSL-1) to the highest
level4. (BSL-4).
At the lowest biosafety level, precautions could include routine hand washing and the use of
minimal protective equipment. Higher degrees of biosafety might necessitate protective
measures including ventilation systems, multiple containment rooms, sealed containers, positive
pressure people suits, documented protocols for all procedures, rigorous staff training, and high
levels of security to prevent access to the facility.
The terms "BSL/1," "BSL/2," "BSL/3," and "BSL/4" are used in the U.S. to denote the basic safe
work practises, specifically constructed facilities, and safety gear required to handle infectious
pathogens. other biological dangers, such poisons. The biosafety level that is highest is BSL/4.
When determining the BSL allocated to a project, the biological risk assessment takes into
account the type of biological hazard, toxin, or infectious agent, including

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1. Its virulence and transmissibility, which affect how contagious it is.
2. The quantity required to spread disease (infectious dose).
3. The range of species that can contract it (host range).
4. It is widely used in the community (epidemiology).
5. The specific lab experiment (activities will perform).
Biosafety Hazards:
Working with well-characterized agents that do not infect healthy people falls under BSL-1, or
biosafety level 1. In general, neither the environment nor lab personnel should be particularly at
danger from these chemicals. In comparison to other levels, this level requires very little
precautions. Staff members of the lab are required to wash their hands before entering and
leaving. Research with these agents can be carried out on standard open laboratory benches
without the requirement for specialised containment equipment. Biosafety level 2 is appropriate
for work involving substances that pose a moderate risk to people and the environment. This
comprises a range of bacteria that can make people slightly ill or that are difficult to inhale via an
aerosol in a laboratory setting. HIV, dangerous strains of E. coli and Staphylococcus,
Salmonella, Plasmodium falciparum, and Toxoplasma gondii are just a few examples. At a
biosafety level 2 or higher, prions can be handled. Prions are infectious agents that spread prion
illnesses like vCJD. Biosafety level 3 should be used for tasks involving germs that might cause
serious and potentially deadly disease when inhaled. These duties could be performed in a
therapeutic, diagnostic, educational, research, or manufacturing environment.
In biosafety level 4 laboratories, diagnostic work and research are conducted on easily
disseminated diseases that might cause deadly disease. These include a number of viruses,
including those that are known to cause Crimean-Congo haemorrhagic fever, Ebola virus, Lassa
virus, and Marburg virus. Other pathogens controlled at BSL-4 include the Nipah virus, the
Hendra virus, and a few flaviviruses. Further, poorly described pathogens that appear to be
closely linked to harmful pathogens are typically handled at this level until sufficient data are

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gathered to either confirm continuation of work at this level or to authorise working with them at
a lower level. Only the Centres for Disease Control and Prevention in Atlanta, Georgia, and the
State Research Centre of Virology and Biotechnology in Koltsovo, Russia conduct research on
the Variola virus, which causes smallpox.

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3.4 Ocular Microscopy
 AIM:
To perform oculo microscopy
 THEORY:
Size is one of the most important physical features employed in the identification and
characterization of an organism. The exact size of a microorganism can only be determined by
utilizing a calibrated ocular micrometer.
An ocular micrometer is a glass disc on which a series of uniformly spaced lines has been
inscribed. The ocular micrometer is placed in one of the eyepieces of the microscope; however,
the distance between the etched lines depends upon the objective lens used to view the specimen.
In order to determine the precise distance between the lines of an ocular micrometer, it must be
calibrated with a stage micrometer . The inscribed lines on a stage micrometer are exactly 0.01
mm (or 10 µm) apart. In order to calibrate the ocular micrometer for a particular objective lens,
the ocular and stage micrometers are superimposed, and the number of ocular graduations per
stage micrometer graduation is determined.
 PROCEDURE:
1. Insert the ocular micrometer into a 10X eyepiece. The ocular micrometer is divided into
ocular divisions(OD).
2. Place the calibrated stage micrometer slide on the stage and focus on the scale. The stage
micrometer has a calibrated scale which is divided into 0.1 millimeters (mm) and 0.01 mm
units.

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3. Adjust the field so the 0 line of the ocular micrometer (OM) scale is exactly superimposed
upon the 0.0 line of the stage micrometer (SM)scale.
4. Without moving the stage micrometer, locate the point as far to the extreme right as possible
where any two lines are exactly superimposed upon each other.
5. Count the number of divisions (mm) on the stage micrometer between the 0.0 line and the
superimposed line to the far right.
6. Count the number of ocular divisions on the ocular micrometer between the 0 line and the
superimposed line to the far right.
7. Divide the distance determined in step 5 by the number of ocular divisions in step 6 and
multiply by 1000 to give the ocular micrometer units in µm.
8. Repeat steps 3 through 7 for each objective on the microscope.
9. If at any time the ocular micrometer is moved to a different microscope or a new objective is
added to the microscope, the calibration procedure must be completed again.
 INFERENCE:
An ocular micrometer makes it very simple to measure the size of micro-organisms that are
mounted on a slide. An ocular micrometer consists of a circular disk of glass which has
graduations engraved on one surface. In some microscopes, the ocular has to be disassembled so
that the disk can be placed on a shelf in the ocular tube between the two lenses; however, in most
microscopes the ocular micrometer is simply inserted into the bottom of the ocular. Before an
ocular micrometer can be used, it is necessary to calibrate it for each of the objectives by using a
stage micrometer. The principle purpose of the exercise is to show you how to calibrate an
ocular micrometer for the various objectives on a microscope.

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PART-4 ( BIO CHEMISTRY )
4.1Chromatography
 AIM:
To perform comparative analysis of two dyes with the help of thin layer chromatography and
Paper chromatography in two different solutions acetic acid and acetone.
 MATERIALS USED:
1. Glass strips
2. Thin paper strips
3. Red ink (Camlin)
4. Blue ink (Chelpark)
5. Acetone
6. Acetic Acid
 Theory:
Chromatography is a technique for separating mixtures that are passed through a media with
distinct moving parts while in a solution or suspension. For the analysis of being samples, we
employ many forms, including paper chromatography and thin layer chromatography. When
samples' RF values are compared, chromatography reveals the polarity of the samples it has
identified.Thin layer chromatography: A thin stationary phase supported by an inert backing is

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employed in this technique to separate the components of a mixture.Paper chromatography:
Using the dissolved chemical compounds' rates of migration over paper sheets, paper
chromatography is a method for separating them.
 Paper chromatography:-
An analytical technique used to separate coloured compounds or molecules is called
paper chromatography. It is mostly utilised as a teaching aid now that alternative
chromatography techniques, including thin-layer chromatography, have taken its place.
 Thin layer chromatography:-
Non-volatile mixtures can be separated using a process called thin layer
chromatography. The experiment is carried out on a piece of glass, plastic, or
aluminium foil that has been lightly covered with an adsorbent substance. Typically,
silica gel, cellulose, or aluminium oxide are utilised as the substance.
 FORMULAE USED:
Entire distance of glass sheet/distance travelled by solution
 OBSERVATION:
1. Using thin layer chromatography, the RF, or the distance travelled by red dye under the
influence of acetone, is 1. 3272365.
2. Using thin layer chromatography, the RF, or the distance travelled by blue dye under the
influence of acetone, is 4.1825723.
3. When utilising paper chromatography, the red dye travelled 64.5 RF, or the distance, under
the influence of acetone. Using paper chromatography, the blue dye travelled a distance of
50.25 under the influence of acetone.
4. Using thin layer chromatography, the RF, or the distance travelled by red dye under the
influence of acetic acid, is 1.3207271.

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5. Using thin layer chromatography, the RF, or the distance travelled by blue dye under the
influence of acetic acid, is 5.14441338.
6. The RF or the distance that red dye has travelled employing paper chromatography is 1.75
for acetic acid.
7. Using paper chromatography, the RF (distance travelled by blue dye under the influence of
acetic acid) is 1.735.
 RESULT:
The greatest RF mean of a red dye analyte determined using paper chromatography is under the
acetone solvent, making it the best method between thin layer chromatography and paper
chromatography.
 CONCLUSION:
Move the absorption caused by reduced polarity, move the distance travelled, or hire the RF
value.
 PRECAUTIONS:
1. The drop applied to the paper must be allowed to dry before the paper is placed in the solvent
to be run.
2. The drop must not be so near the PAGE 103bottom of the paper that it is immersed in the
solvent.
3. The paper must not touch the sides of the test-tube except at its four corners.

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4.2Chlorophyll Extraction
 AIM:
To extract chlorophyll from plant sample and estimate chlorophyll content.
 THEORY:
According the endosymbiotic hypothesis, a eukaryotic cell engulf a cyanobacteria which survive
inside a cell and later on evolve to become chloroplast. Chloroplast are membrane bound
organelle found in algae and plants which perform photosynthesis .Plant chloroplast are lens
shaped usually 5-10 um in diameter and 1-10 um thick. Chloroplast is one of the characteristic
organelle of plant kingdom commonly found in leaves. The main function of chloroplast is to use
light (natural light) to perform photosynthesis . Chloroplast contains a special pigment known as
chlorophyll .The green colour of leaf is due to the presence of chlorophyll .Usually to study the
different steps of photosynthesis effect of photosynthesis modulator, we need to extract/ isolate
chloroplast .
 MATERIAL REQUIRED:
Mixture grinder , Test Tubes , Distilled water , MicroPipettes, Beakers, scissors, measuring
tubes , sucrose, Green leaf samples or other greenish plant tissue samples, MgO or MgCO3,
100% or 80% (v/v) acetone or diethyl ether, spectrophotometric grade, Hydrophobic organic
solvent: diethyl ether, light petrol, or hexane, spectrophotometric grade, Half-saturated NaCl
solution, Anhydrous Na2SO4,Mortar and pestle Aluminum dishes, 100°C drying oven, Freeze

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dryer, 5-ml graduated centrifuge tubes, Explosion-proof tabletop centrifuge or a cooling tabletop
centrifuge, UV-VIS spectrophotometer, 1-cm-path-length cuvette, 25- or 50-ml separatory
funnel, Water bath set below 35°C (optional)
 PRINCIPLE:
In the laboratory, the cells of the leaves are disrupted the untethered organelles which can then
be sorted out from each other by filtration and different centrifugation as cell fraction .Filtration
will remove cell debris (walls) and unbroken cells providing a filtrate that certain organelles,
chloroplasm, nuclei, mitochondria, ribosomes . Small membrane vesicles and soluble
components. Most of these will not be visible under microscope .Low speed centrifugation will
sediment remaining last bodies and moderate speed centrifugation will sediment chloroplast,
living mitochondria and ribosomes and other soluble components in the supernatant .
 PROCEDURE:
1. Obtain 8g of divined leaf tissues cut into pieces and 1cm2.
2. Place leaf tissues in a pre-chilled blender cup containing 40 ml ice cold 0.5 m sucrose
blend for 20s at top speed .
3. Remove the blended contain and squeeze the 4 layers of pre chilled cheese cloth/ muslin
cloth into the cold beaker by twisting the top corners around each other.
4. Pour 14 ml of the homogenate in each of the centrifuge tube and centrifuge for 200g for
5min.
5. Using a pastured pipette, transfer each supernatant(containing chlorophyll) to another
tube and centrifuge for 1000g for 7min
6. Using the pipette, discard the supernatant but be careful not to disturb the palette.
7. Pour 2ml of phosphate buffer on palette and gently suspend it up and down using a clean
pastured pipette and buffer the volume make up to 8 ml

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8. Estimation of chlorophyll A conc of the suspention –
i. Measure 4.75 ml of ice chilled 80% acetone and take it in test tube .
ii. Add 0.25 ml/ 250 ul of chlorophyll suspension, mix well and read the absorbance
at 652 nm against 80% acetone.
9. Determination of chloroplast concentration of the suspension-
Measure 4.75 ml of the buffer in a test tube, add 0.25 ml of chlorophyll suspension and
mix it well
 INFERENCE:
In this experiment , we successfully extracted chlorophyll from the chloroplast of leaves and
counted the number of chloroplast present under the microscope .
 PRECAUTIONS:
1. Leaves should be properly deveined.
2. Over heating or over grinding of leaves can lysis the plastids and chlorophyll will not be
extracted in the experiment in that case .
3. Handle the equipment carefully.
4. Clean the spectrometer before use.

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PART-5 ( BIOINFORMATICS )
5.1 Database Searching from NCBI for Data Retrieval FASTA
Analysis
 AIM:
To prepare a NCBI report on a protein using NCBI.
 TOOL USED:
NCBI
 THEORY:
A Canadian programme sought to engage front-line health professionals in continuously
improving the services they offered to people with chronic conditions.29
A programme theory-
driven evaluation approach was used to describe the processes that might lead to the programme
outcomes, and the conditions under which these processes were believed to operate. Using
multiple sources—literature review, programme documents, committee meetings, observations,
focus groups and interviews—a programme theory was developed. This took the form of a
diagram (figure 1) and a narrative account of the theoretical basis of the intervention. The

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narrative identified relevant mid-range theories—including work motivation theory and
reflective learning—as well as the importance of dissonance between actual behaviour, pursued
goals and outcomes. It showed the relevance of these to components of the intervention, which
included feedback, reflective learning and action planning.
The articulation of this theory helps in characterising the programme components, the mediating
processes through which they work and the moderating factors related to participants and
contexts, as well as improving the ability to measure the intervention's impacts on practice
change.
 PROCEDURE:
1. Search NCBI on any browser and open the first link website (official website of NCBI).

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2. Go on the search column in the website layout and search for “Thymosin AND Galleria
mellonella” by selecting option of nucleotide.

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3. Then after many files or reports to get data select one.

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4. After selecting one report click on the FASTA option appearing in the top left side click it.
5. Then after this part repeat the 2, 3 and 4 step by clicking the option protein in the first
website layout page.
6. After this we are able to get the code of our desired protein.
 OBSERVATION/SCREENSHOT:
The FASTA nucleotide sequence of Thymosin found in Galleria mellonella is:

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The FASTA protein sequence of Thymosin found in Galleria mellonella is:

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 RESULT:
The FASTA nucleotide sequence of Thymosin found in Galleria mellonella is:
>MG846901.1 Galleria mellonella thymosin (TMS) mRNA, complete cds
CCTGCTTGCCGGCCGACTTCGCTCAGACCGCACGTTCACTTCAGTCGCGTTTTGAGAGTGATAGGTCGTG
TACGTTTTATCGTATATCCCCTTCCTGCATTTTATAACTTGATCTAAAATGGCCTGCTCAGTGAGTGACA
CCCCCTCCCTGAAGGATCTGCCTAAAGTGGCCACAGACCTCAAGAGCCAGCTCGAGGGATTCAACACGAG
TTGCTTGCGCGATGTTGACACCAACGAGAAGATCGTGCTTCCGTCTGCTGAAGATGTTGCCACAGAGAAA
AACCAAAAGTCCTTGTTCGACGGCATTGAAAAATTCGACGCTAGCAGGTTGAAGCACACAGAAACCCAGG
AGAAAAATCCACTGCCAGACAAAGATGTTGTAGCAGCGGAGAAGGCACACCAGAATCTCCTTGACGGCGT
GGAACATTTTGACAAGACACAGATGAAACATGCAACGACCGAAGAAAAGAACCCCTTGCCGCCCATTGAA
GCGATCGAAGCTGAGAAGGAGAAGAATAAATTCTTAAACGGCATCGAGAACTTCGACCCGAGCAAGCTGA
AGCACACCGAGACTTGCGAGAAGAACCCGCTGCCCACCAAGGACGTCATTGAACAGGAGAAGACGGCTTG
AACGTCCGCATCCATTGCCCGCAATTATATCTCGCTAATATCGCCGTATTTTAAACTCGATGTAAGTTAA
TGACCCCGTTTGCACTGTCCTATCAAAACGTCAATAAATACTCATCAGGGTTTCTTTCAAGTTCTTTTTT
TTAATTTTCAAAATCTTTAGAAAGGAATGGTTATACCATACGTCTACTATATTTCTTTCTTTTTTTTTCC
CTTTTCTATATTATTAAGAAGGATTTTATTGTTCCAATACGCACGTGGAATTGACGATTTGTCGGCGCAA
GATAAAGATGCGGTTCGTGAAAAAGATATAATAGAAATAAGTTCACTTTTACGGAATGAAACCCTGATTT
TACACCATCTGGCCTAAGTCATAAGCGAAGAGGACTGCAAACTGGCTCTTATCATTAGTTTATAGGAACC
AAATTTCTATGTCACTGTAACGTATATATTTTAATAAGTGTATTTTATGAGCATTTTATAATTTAATTTA
TATTATATTGAGACGGATAATTAAGTATTAATGATATT CAGTACTGATTTGTGTAGGCTGAGAACACTAC
AATTTGAAATTTGTTTTGTACTCTTTCGTTTTGTGCTTGTGTTCTCTATTTAGTTCGAGGTATATTAAGT
ATTTACGAGATCGCCTGTAGGTATGTACGTGATGCCTCTGGTCAATGAATAAATTAGAAGTGTAGTTACC
AAAACACAATTTGGTTCTAAATTATTGTACCGTGATTGTAATTTCTCTATTGATATTTTATTATGCTGTA
AAGTAATGTCATAATGTCGACAGAGAAACGAATGAAAAAAGAAAAAATTAAAAATGACAGTAAGTACTCT
AGTAATATTACTTGCTCAACTGTGTACGAAATAGATAAGGTTTGGTCTGATTTTTAAAAAAGCAATAGTT
TGTTTAGTTTGATAAAATAGTGTATAACAATAATATTTTATATTGTCATATATAAATCAATAATTTTCCA
ATTTTATAATATGCTATCGATGCCAATACTTGAAACATTTTTACCGGAATTTTGTATGTTTTACAGACGG
TGCTTTTTTATAAATATAGATTGAAA

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>AXY94753.1 thymosin [Galleria mellonella]
MACSVSDTPSLKDLPKVATDLKSQLEGFNTSCLRDVDTNEKIVLPSAEDVATEKNQKSLFDGIEKFDASR
LKHTETQEKNPLPDKDVVAAEKAHQNLLDGVEHFDKTQMKHATTEEKNPLPPIEAIEAEKEKNKFLNGIE
NFDPSKLKHTETCEKNPLPTKDVIEQEKTA
 LEARNING OUTCOMES:
1. The protein and nucleotide sequence of Thymosin.
2. Getting data from NCBI.
3. How to use this data for researches.
 REFRENCES:
https://www.ncbi.nlm.nih.gov/protein/AXY94753.1
 PRECAUTIONS:
1. Type Boolean word AND in upper case only.
2. Do not use partial proteins for the full format study.
3. Copy the code sequence properly for study.

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5.2 Database Searching from NCBI for Data Retrieval BLAST
Analysis
 AIM:
To prepare a NCBI BLAST report on a protein and mapping its evolution tree.
 TOOL USED:
NCBI, NCBI BLAST
 THEORY:
The proper sampling of homologous series for phylogenetic of molecular evolution evaluation is
an important step, the high-satisfactory of that can have a vast effect at the very last
interpretation of this study. There isn't any unmarried manner for building datasets appropriate
for phylogenetic evaluation, due to the fact this undertaking in detail relies upon at the medical
query we need to address, Moreover, BLAST - Explorer offers an easy intuitive and interactive
graphical illustration of the BLAST consequences and lets in choice and retrieving of the
BLAST hit sequences primarily based totally a huge variety of criterions . This gear offers a
phylogenetic orientated graphical show of the BLAST result.
 PROCEDURE:

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1. Search NCBI on any browser and open the first link website (official website of
NCBI).
2. Go on the search column in the website layout and search for “Thymosin AND
Galleria mellonella” by selecting option of nucleotide.

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4. After selecting one report click on the FASTA option appearing in the top left side
click it and copy the FASTA format code of the protein.
5. Then search “NCBI BLAST” in a new tab and open the first site appearing on the
page.

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6. Click on the option protein blast having this type of output site after clicking. After
this paste the amino acid sequence in the column asking for FASTA sequence or
accession number (can also upload the txt file having the FASTA format code of
protein) and give a desired title to the file.
7. Scroll down and go the database part and select the option “UniProtKB/Swiss-
Prot(swissprot)” option and finally click the option BLAST in the bottom-left corner.
8. A new web page is appeared having the Blast report and by this report we find the
evolution table and know which organism is related by protein blast.

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9. Click on the option in the top-right named “Distance tree of results”after In this page
change the tree method by neighbour method and the final evolution graph of

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Thymosin appeared.
 OBSERVATION/SCREENSHOT:

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Report of BLAST file:
Evolution tree:
 RESULT:
FASTA protein sequence of Thymosin found in Galleria mellonella is:

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Evolution graph of thymosin is:
4. How to compare two amino acid code using NCBI BLAST.
5. How to plot an evolution tree of a protein.
 PRECAUTIONS:
4. In the search option paste the protein sequence properly.

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5. After starting BLAST let it for few seconds till the full report appears.
 REFRENCES:
https://www.ncbi.nlm.nih.gov/protein/AXY94753.1 for amino acid code
https://blast.ncbi.nlm.nih.gov/blast/treeview/treeView.cgi?request=page&blastRID
=CBNVA3XA01R&queryID=lcl|Query_710726&entrezLim=&ex=&exl=&exh=& ns=100 for
blast evolution tree.

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5.3 Retrieving Data of Five Same Species for Multiple Genome
Sequencing to Get their Phylogenetic Relationship
 AIM:
To perform the comparative analysis of a same protein found in different organisms and potting
an evolution tree for ancestral analysis.
 TOOL USED:
NCBI, NCBI BLAST, EBI, RCSB, Notepad
 THEORY:
Amino acid sequencing is the process of identifying the arrangement of amino acids in proteins
and peptides . Each protein or peptide consists of a linear sequence of amino acids . The protein
primary structure conventionally begins at the amino-terminal (N) end and continues until the
carboxyl terminal (C) end . The structure of a protein may be directly sequenced or inferred from
the sequence of DNA . The amino acid sequence of a protein or peptide is useful information to
understand the protein or peptide , identify it in a sample and categorize its post translational
modifications . The process of determining the amino acid sequence is known as protein
sequences .
 PROCEDURE:

DESIGNED BY: ISHANT
GUPTA
mellonella” by selecting option of protein.

DESIGNED BY: ISHANT
GUPTA
5. Then search “NCBI BLAST” in a new tab and open the first site appearing on the page.
6. Click on the option protein blast having this type of output site after clicking. After this paste
the amino acid sequence in the column asking for FASTA sequence or accession number
(can also upload the txt file having the FASTA format code of protein) and give a desired
title to the file.
7. Scroll down and go the database part and select the option “UniProtKB/Swiss-
Prot(swissprot)” option and finally click the option BLAST in the bottom-left corner.
8. A new web page is appeared having the Blast report. From this report we know which
species organism is related having same protein structure and search for FASTA sequence of
thymosin.

DESIGNED BY: ISHANT
GUPTA
9. Pick up top three or more species for the comparative analysis of the amino acids and search
their FASTA sequence and paste them into a notepad like this.
10. Then search “multiple sequence alignment” and open the first site page and in that page
select the option “launch kalign”.

DESIGNED BY: ISHANT
GUPTA
11. Paste the whole notepad copied sequence in the sequence part or uploads the doc file in it and
click the option submit in the bottom of the page

DESIGNED BY: ISHANT
GUPTA
12. A new web page appears having output of comparative analysis.

DESIGNED BY: ISHANT
GUPTA
13. You can also read different type of data like ancesteral tree.
 RESULT:
Phylogenetic tree for ancestral analysis of Thymosin protein is:

DESIGNED BY: ISHANT
GUPTA
4. Know about the ancestral analysis of same protein.
5. Know about the similarity of structures of same protein between different organisms.
 PRECAUTIONS:
6. Do not upload file and paste the five sequences of organisms simultaneously in the multiple
genome sequencing part.

DESIGNED BY: ISHANT
GUPTA
 REFRENCES:
https://www.ncbi.nlm.nih.gov/protein/AXY94753.1 For the protein sequence
https://blast.ncbi.nlm.nih.gov/Blast.cgi For the blast report
https://www.ebi.ac.uk/Tools/services/web/toolresult.ebi?jobId=kalign-I20220710-153653-0978-
15639139-p1m&analysis=phylotree For the multiple sequencing report.
https://www.rcsb.org/3d-view/1HJ0 For 3-D analysis of protein structure.

DESIGNED BY: ISHANT
GUPTA
5.4 To Retrieve a Protein Sequence from POB Database and
Analyze its 3D Structure
 AIM:
To Retrieve a Protein Sequence from POB Database and Analyze its 3D Structure
 TOOL USED:
NCBI, NCBI BLAST, EBI, RCSB, Notepad
 THEORY:
A protein’s biological function is dictated by the arrangement of the atoms in the three
dimensional structure . This could be the arrangement of catalytic residues and an active site or
how a protein interacts with other proteins for structural or other regulatory purposes . Having a
protein structure provides a greater level of understanding of how are proteins works which can
allow us to create hypothesis about how to affect it , control it or modify it . The protein
databank is a database for the three dimensional structural data of large biological molecules
such as proteins and nucleic acids . The data , typically obtained by X ray crystallography , NMR
spectroscopy or increasingly cryo-electron microscopy and submitted by biologists and
biochemists from around the world .
 PROCEDURE:

DESIGNED BY: ISHANT
GUPTA
2.
mellonella” by selecting option of protein.

DESIGNED BY: ISHANT
GUPTA
4.
6.

DESIGNED BY: ISHANT
GUPTA
8. Then search “NCBI BLAST” in a new tab and open the first site appearing on the page.
9. Click on the option protein blast having this type of output site after clicking. After this paste the
amino acid sequence in the column asking for FASTA sequence or accession number (can also
upload the txt file having the FASTA format code of protein) and give a desired title to the file.
10. Scroll down and go the database part and select the option “UniProtKB/Swiss-Prot(swissprot)”
option and finally click the option BLAST in the bottom-left corner.
11. A new web page is appeared having the Blast report. From this report we know which species
organism is related having same protein structure and search for FASTA sequence of thymosin.

DESIGNED BY: ISHANT
GUPTA
12. Pick up top three or more species for the comparative analysis of the amino acids and search
their FASTA sequence and paste them into a notepad like this.

DESIGNED BY: ISHANT
GUPTA
13. Then search “multiple sequence alignment” and open the first site page and in that page select
the option “launch kalign”.
14. Paste the whole notepad copied sequence in the sequence part or uploads the doc file in it and
click the option submit in the bottom of the page

DESIGNED BY: ISHANT
GUPTA
15. A new web page appears having output of comparative analysis.

DESIGNED BY: ISHANT
GUPTA
16. You can also read different type of data like ancesteral tree.
17. Now search RCSB on google in a new tab and click on the first link appearing on the page
having layout like this. And write “Thymosin” in the search box and search it.

DESIGNED BY: ISHANT
GUPTA
18. Select a data from the group for your study and open it .
19. Click on 3D View appearing on the top of the page. A new web page opens having the 3D
structre of the protein.
From this whole data you can study about the shape, size, structure, work action and many more
things about the protein.

DESIGNED BY: ISHANT
GUPTA
 RESULT:
3D Structure of Thymosin found in Bos Taurus is:
4. Know about the ancestral analysis of same protein.
5. Know about the similarity of structures of same protein between different organisms.

DESIGNED BY: ISHANT
GUPTA
6. We can know about the bonding structure of the protein weather is α-helix or β-plated.
7. We can know about the components of the protein for making their antidotes or transcript it.
 PRECAUTIONS:
6. Do not upload file and paste the five sequences of organisms simultaneously in the multiple
genome sequencing part.
 REFRENCES:
https://www.ncbi.nlm.nih.gov/protein/AXY94753.1 For the protein sequence
https://blast.ncbi.nlm.nih.gov/Blast.cgi For the blast report
https://www.ebi.ac.uk/Tools/services/web/toolresult.ebi?jobId=kalign-I20220710-153653-0978-
15639139-p1m&analysis=phylotree for the multiple sequencing report.
https://www.rcsb.org/3d-view/1HJ0 For 3-D analysis of protein structure.

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