Yashwantrao
Maharashtra
Chavan
Open University
Post Graduate Degree Programme (Bio-Technology)
SBT095: Lab Course M. Sc. (Bio-Technology)
Semester 9 Lab
Workbook
1
Yashwantrao Chavan Maharashtra Open University
Vice-Chancellor-Dr. Rajan Welukar
Expert Advisory Committee
Mr. Manoj Killedar
Director, School of Science & Technology, Y.C.M. Open University, Nashik
Mrs. Sunanda More
School of Science & Technology, Y.C.M. Open University, Nashik
Mrs. Chetana Kamlaskar
School of Science & Technology, Y.C.M. Open University, Nashik
Dr. Sunil Ganatra
135, Krushnakunj, Toata Colony, Lakadganj, Nagpur
Prof. Indira Ghosh
Bio-Informatics Center, University of Pune, Pune
Prof. Urmila Kulkarni-Kale
Bio-Informatics Center, University of Pune, Pune
Prof. Dr. Piyali Kar
Maharashtra Education Foundation, Foundation Towers, Sector – 11/20, CBD Belapur,
Navi Mumbai
Course Writer
Mr. Pravinkumar Domde
G.H. Raisoni Institute of Interdisciplinery Sciences, Sharadha House, 345,
Kingsway, Nagpur
Course Editor
Dr. Suchitra Godbole
G.H. Raisoni Institute of Interdisciplinery Sciences, Sharadha House, 345,
Kingsway, Nagpur
Course Coordinator and IT Editor
Mrs. Sunanda More
School of Science & Technology, Y.C.M. Open University, Nashik
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Manoj Killedar, Director, School of Architecture, Science & Technology
E-Version available at
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© Yashwantrao Chavan Maharashtra Open University, Nashik
Printed & Published by: Shri. S.P. Kowale, Registrar, Y.C.M. Open University, Nashik
2
CERTIFICATE
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is
to
certify
that
…………………………………………………………………………….
Mr./Mrs./Smt.
has successfully completed experiment as per the following details:
(1) Total number of experiment completed: ……………….…………………
(2) Serial number of experiment completed: …………………………………
Laboratory Instructor Name : ……………………………………….…………………….
Signature & date
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3
List of practical of M.Sc. Biotechnology Course SBT 095
Sr No. Titles of Practical Activity
1
2
3
4
Preparation of Antigen
Immunization of Mice using different kinds of antigens
Separation of serum proteins by agarose gel electrophoresis
To perform immunoelectrophoresis (IE) using an antigen-antibody
system
5
To perform Rocket – Immunoelectrophoresis or Electrophoresis in
Antibody- containing media
6
7
Study of immunoprecipitation - Quantitative Precipitation Test
Precipitation in gel - Single Radial Immunodiffusion (SRID)
8
9
10
11
12
13
Precipitation in gel - Double diffusion assay
To perform Enzyme Linked Immunosorbent Assay (ELISA).
To perform western blot analysis.
Purification of Immunoglobulin
Preparation of lymphocytes
Agglutination reaction with reference to blood grouping and Blood
examination for Rh factor
14
15
16
17
18
19
Widal test using tube agglutination reaction
Isolation of DNA from Blood Cells.
Isolation of RNA from yeast.
Restriction endonuclease digestion of genomic DNA.
Analysis of DNA fragments by agarose gel elctrophoresis
Isolation of Plasmid DNA from bacteria by alkaline lysis miniprep
method.
20
21
22
23
Preparation of competent cells
Transformation of bacterial cell with foreign DNA
Filing of Patent with example
Patent search on internet
4
Experiment No. 1
Aim: Preparation of Antigen
To prepare Antigen Soluble- BSA, insoluble - sheep RBC and
particulate (Whole organism) - Pathogen
Principle
Antigen is defined as the agent, which when introduced into the body
reacts or binds with the products of the immune response (e.g.
antibody).
Immunogen is defined as the agent which when introduced into the
body can not only reacts or binds with the products of the immune
response but also induces and immune response.
Characteristics of an Immunogen
1. Molecular weight: Generally high molecular weight molecule can
act as immunogen e.g – BSA (66 K Da), Ovalbumin (42 KDa).
low molecular weight molecules when coupled with a carrier BSA
acts as an immunogen. Such low molecular weight molecules are
called Haptens.
2. The immunogen would be a foreign substance so that it is not
recognized as ‘self’ by the host organism.
3. Chemical Structure: To induce a strong immune response
against a particular antigen, it is required that antigen has the
capability to induce more than one type of antibodies. If for
example a polypeptide with homopolymer structure is injected, it
will only trigger one type of antibody. Thus a weak response will
be seen.
4. Degradability: The immunogen should be stable for a sufficient
time, enough to induce a strong antibody reaction. If the foreign
molecule is degraded very fast then enough antibodies will not
develop so as to be detected easily.
Classification of Antigens
Based on the source from which an antigen originally originates, they
are classified in the following way:
1 Natural Antigen: RBC, WBC
2 Exogenous: Pollen, Microbes
3 Endogenous: Within the host e.g. Mitochondrian etc.
4 Artificial: Hapten
Among these categories various types of antigens can be listed:
5
A Soluble Antigen: BSA
B Insolubel Antigen: Cells (RBC), pollen
C Whole organism: Bacteria
Procedure
A. Soluble Antigen BSA
Quantity to be prepared : 50ml
Amount of BSA per ml : 0.5 ml
To be prepared in PBS ( Phosphate buffered saline)
Preparation of PBS
NaCl
: 8 gm
KCl
: 0.2 gm
KH2PO4
: 0.2 gm
Na2HPO4.2H2O : 1.15 gm
Water
: 1000ml
: 7.2
PH
Prepare 50 ml. To this add 25 mg of BSA
B. Sheep RBC
Anticoagulant Solution ( Alsever’s solution)
Quantity to be prepared: 100ml
Components
Dextrose
Trisodium Citrate
NaCl
Citric acid
Water
: 20.5 gm
: 8 gm
: 42.0 gm
: 0.55 gm
: 100ml
RBC
1. Collect blood (Sheep’s) from jugular vein with the help of a
sterilized syringe.
2. Add Alsever’s solution (1ml to every 5 ml of blood) and shake.
3. Spin at 2000 rpm at room temperature for 5 min.
4. Discard supernatant and resuspend pellet in PBS.
5. Spin at 2000 rpm for 10 min at room temperature.
6. Suspend in PBS. For long time storage add 1% sodium azide.
C. Whole Organism
1. Use any microbe at a concentration of 106 cells per ml.
2. Heat inactivate by keeping at 65oC for 10 min. Or treat with 5%
formaldehyde.
6
Experiment No. 2
Aim: Immunization of Mice using different kinds of antigens
Principle
The Immune Response
An immune response is consequence of a complex sequence of events
involving antigen and at least three kinds of lymphoid or
reticuloendothelial cells. Most antigens require ‘T’ cell help to stimulate
‘B’ cells into antibody production in response to antigen (foreign)
present in the blood. A further complexity is introduced into the
system by the different classes (isotopes) of antibody molecules
produced by B cells and the past immunization history of the animal
which is reflected by the number of B memory cells present. A high
number of memory cells result in a very large and much more rapid
response to a second injection ( Booster Dose) of antigen – a
secondary response – a classical feature of the immune response.
Parameters Affecting Immune Response
Antigen Dose: It is clear that the nature of the antigen introduce
parenterally into an animal is important; however the amount is of
even greater significance. Too little or too much antigen can be
striking counter-productive from the point of view of the immunologist
trying to obtain a maximum yield of antibody. Optimum dosage for
various experimental animals is as follows:
Animal
Dosage
Mice
Rabbit
Rats
Guniea Pigs
Sheep or Goat
Chicken
: 5 – 50 µg
: 50 - 500 µg
: 10 – 100 µg
: 10 – 100 µg
: 0.5 – 200 µg
: 50 – 200 µg
Age of the animal: Immune response is strikingly age dependent.
New born mammals having next to no innate ability to synthesize
antibody and old animals showing a response very significantly
reduced by comparison with that seen in their prime maternal antibody
is transferred passively either across the placenta or orally thorough
colostrum of milk: such passively acquired antibody can interfere with
the process of active immunization.
7
Genetic factor: Certain antigens (Ag) can elicit an immune response
in inbred strains but not in others. This ability to respond to the
antigen has in many cases been shown to be under genetic control of
loci closely linked to a part of the MHC.
Similar genetic factors are undoubtedly a major source of
variation in an immune response when out-bred animals are
immunized.
General Health: The general health of the animal being used to raise
an antiserum can have a significant effect on antibody (Ab) levels.
Poor nutrition, disease ridden animals or dirty housing and other stress
situations can be reflected in poor response.
Rout of immunization: There are five routes through which antigen
can be injected.
1. Intramuscular
2. Intravenous
3. Intradermal
4. Intraperitoneal
5. Subcutaneous
Specific routes are taken depending on the animal to be immunized
and the type of antigen used. Intravenous injections are not given if
the antigen is particulate matter because otherwise it may cause
cross-reaction. Intraperitoneal injections are given to small animals
like mice, rats etc.
Antigen and Adjuvants
The most important factor to be considered in immunization
experiments is the type of antigens used. It is very obvious that an
antigen should be an immunogen also i.e. it should spark off an
immune response. This having been taken care of, antigen is chosen
from antigen classes of proteins, polysaccharides, synthetic
polypeptides, nucleic acid, chemically modified antigens, low molecular
weight substances etc. All these vary in efficiency and speed of
instigating an immune response.
An essential required condition for immunization is that the
immunogen should degrade slowly in the body of experimental animal.
This to make sure that immune system of the animal gets ample time
to synthesize Ab against the Ag. To make the Ag last longer in the
body adjutants are used. Various types of adjutants used these days
are:
1. Freund’s Complete Adjuvant (FCA):- Its components are
8
2.
3.
4.
5.
6.
7.
8.
a) Mineral oil Bayol – f3
b) Emulsifying agent mannide monooleate
c) Muramyl dipeptide components of the membrane of heat
killed mycobacterium
Freund’s incomplete adjuvant (FIA): it is FCA only but without
the bacterial component
Insoluble aluminium salt + calcium
Heat killed Mycobacterium tuberculosis
Bacterial lipopolysaccharide.
Saponin.
Synthetic polynucleotide
heat killed Bordetella pertusis
Saponin is safer. Used in veterinary medication
Raising antibodies in Mice and Rabbit
Raising antibodies in mice
Mice: Depending on the strain of mice used, the Ab production
varies. For example:
BALB / C
Intermediate
CS7BL / 6
Low
CBA
High
Use BALB / C or Swiss strain of mice.
Ab Production
Ab Production
Ab Production
Mouse Handling
1. Remove the mouse from cage by gently grasping the tail (with
preferred hand ) at the base
2. Place the animal on a wire-bar cage lid to permit grasping.
3. Approach the back of the neck from the rear with free hand.
Firmly grasp the skin behind the ears with the thumb and the
index finger.
4. Transfer the tail from preferred hand to beneath the little finger
of the hand holding the skin of the neck.
5. Observe or inject the restrained mouse.
9
Animal Identification
Proper identification of animals plays an important role in
research, whether animals are being used as experimental subjects or
for breeding purposes, or as a source of tissues, cells or fluids.
Basic information is usually maintained on cage cards which
should be utilized to identify single or group housed rodents where
individual identification is not necessary. In some cases it is necessary
to individually identify rodents.
Usually for mice ear-punching is used for identification and for
rabbits and goats ear tagging with numbered metal clips are used.
Raising antibodies in mice
Antigen: sheep RBC (25%)
1. Inject 100 ul of 25% sRBC (sheep RBC) to each animal either
subcutaneously or intraperitoneally.
2. Label the animal by ear puncture and label also the cage.
Protocol for injection
1 Inject 100 µl of 25% sRBC on day one through intraperitoneal route.
2 Give booster injection ( 100 µl, 25% sRBC) on day 15 through
subcutaneous route.
Bleed after 3 days by retroorbital bleeding / heart puncture.
Raising antibodies in rabbit
Antigen: BSA
1. Take freund’s complete adjuvant in small flask (1.5ml) or in
microfuge tube also
2. To this add antigen BSA (also 1.5 ml, 0.5 mg per ml) drop by
drop and vortex simultaneously.
3. Mix adjuvant thoroughly before adding. This is done to uniformly
distribute mycobacteria.
4. Inject one ml into rabbit subcutaneously.
5. Lable the animal.
Protocol for injection
1 first injection on 1st day – 1 mg/ml of BSA – subcutaneous injection
with complete adjuvant.
2. Give second injection on day 7, 500 µg / ml with incomplete
adjuvant.
10
3. Booster injection can be given on day 28th and 29th. 500µg/ml with
incomplete adjuvant
Bleed after 5 days by ear bleeding/ heart puncture.
Negative Control
Maintain negative control by exposing another set of animals to some
conditions i.e., wounding with needle and injecting phosphate buffered
saline without antigen.
Collect preimmune serum for all animals before immunization by
ear bleeding the rabbit and tail bleeding the mice.
Observation
Observe the immunized and control animals for the development of
antibodies against the antigen used for immunization.
Result
The given animal ---------- has been immunized with the ------------antigen successfully.
11
Experiment No. 3
Aim:
Separation
electrophoresis
of
serum
proteins
by
agarose
gel
Principle
Studying antigen antibody interaction solely by simple diffusion is
possible if there are only a few components in the system but, if there
are multiple antigens with several antibodies, the precipitin lines
become difficult to resolve and impossible to interpret.
Increased
resolution
can
be
obtained
by
combining
electrophoresis with immunodiffusion in gels, in the technique known
as immunoelectrophoresis. This is useful in the immunological
examination of serum protein: Serum proteins separate in agar gels,
under the influence of an electric field, into an albumin and α1-, α2 -,
β – and γ- globulins.
Agarose gel electrophoresis
Materials and Equipments
2% agar in barbitone buffer
Barbitone buffer
Precoated microscope slides
Normal and myeloma sera
10% v/ v glacial acetic acid in water
Electrophorsesis tank and power pack
Gel punch
Barbitone Buffer, pH 8.2, Ionic strength 0.08
Materials
Barbital sodium (5’5-diethyl barbituric acic, Na salt)
Barbital (5’5 diethylbarbituric acid)
5M sodium hydroxide
Merthiolate
Method
1. Dissolve 12.00g sodium barbital in 800 ml distilled water.
2. dissolve 4.40 g barbital in 150 ml distilled water at 95oC.
3. Mix solutions 1 and 2 and adjust pH to 8.2 with concentrated
sodium hydroxide.
4. Add 0.15 g merthiolate ( preservative) and adjust the final
volume to 1000 ml.
12
Method
1. Melt the agar in a microwave oven/ on a hotplate.
2. Mark the end of the slide that will be positive during the
electrophoresis. If required number the slide.
3. Pour 3-5 ml of agar onto the slide on a leveled surface.
4. When the agar has set, punch the pattern. (Smaller wells than
used for immunodiffusion are required. A fine Pasture pipette or
a hypodermic needle with a square cut end may be used.)
5. Suck out the agar plugs.
6. Fill the wells with the serum to which small amount of
bromophenol blue dye had been added
7. Fill the electrophoresis thank with full – strength barbitone
buffer.
8. Place the slide in the electrophoresis tank and connect each end
of the slide to the buffer chambers with rayon or filter paper
wicks. Close the tank.
9. Apply a current of about 8 mV/ slide.
Note: The bromophenol blue dye binds to the serum albumin and as
this is the fastest migrating band it serves as a marker through out the
electrophoresis. If excess dye has been added, however, a bright blue
band of free dye will run in front of the albuim towards the anode.
10.
When the albumin band reaches near the end of the slideafter about 60 min – remove the slide and fix the proteins by
immersing the slide in 10 % glacial acetic acid.
11.
Cover the slide with fine filter paper and leave for drying.
12.
Dampen the paper and remove, then stain the slide with
Coomassie brilliant blue.
Suggested design
One well should contain normal serum and the other serum from a
patient with multiple myelomatosis ( a disease in which a single clone
of antibody – forming cells becomes malignant and produces large
amount of antibody). If you are using mouse reagents, then ascitic
fluid or serum from a hybridoma – bearing animal will do equally well.
Results
The main serum proteins should show clearly as oval bands. Identify
each band
(albumin, α1-, α2 -, β – and γ – globulins ), and
assign the abnormal monoclonal band to one of these.
13
Experiment No. 4
Aim: To perform immunoelectrophoresis (IE) using an antigenantibody system.
Principle
This is a powerful analytical technique with great resolving power.
Immunoelectrophoresis
combines
separation
of
protein
by
electrophoresis with identification by double immunodiffusion. An
antigen mixture is first electrophoresed and separated by charge
direction of the electric field and antiserum is added to the trough. The
formation of precipitation band with polyvalent or specific antiserum
identifies individual antigen components. IE is widely used to in clinical
labs to detect the presence or absence of protein in the serum. The
technique is applied for the antigen which migrates to the positive
pole. It is used to detect immunodeficiency diseases.
Materials and equipment:
As for agar gel electrophoresis but in addition:
Anti- human whole serum
Method
1. Prepare slide as for agar gel electrophoresis.
2. Cut the pattern shown in figure below. ( Although cutters and mould
are available commercially for many different patterns, the holes can
be made with hypodermic needles ( cut square and sharpened) and
the trough with razor blades.)
3. Suck out the agar wells but do not remove the agar from the trough
as this may cause abnormalities in protein banding during
electrophoresis.
4. Fill one well with normal human serum and the other with myeloma
serum.
5.run the Electrophoresis as before.
6. Remove the agar trough and fill with anti-whole human serum.
7. Leave the slide to incubate overnight in a humid chamber at a
constant temperature.
(again lines will appear within 2-3 h if the slide is incubated at
37oC.)
8. Examine the lines produced and identify the Ig G, Ig A and Ig M
bands, and the bump in the precipitation arc typical of monoclonal
immunoglobulins in the myeloma serum.
14
Observation
From the figures and photos displayed below observe and
examine the lines produced and identify the Ig G, Ig A and Ig M bands,
and the bump in the precipitation arc typical of monoclonal
immunoglobulins in the myeloma serum.
Figures and photographs showing electrophoretic pattern of
serum immunoglobulins
15
Result
Separation of immunoglobulin by Immunoelectrophorsis of given
sample has been performed successfully and found to contain
--------------------------
16
Experiment No. 5
Aim: To perform Rocket – Immunoelectrophoresis
Electrophoresis in Antibody- containing media
or
Principle
In this technique negatively charged antigen is electrophoresed in a
gel-containing antibody. The precipitate formed between an antigen
and antibody has the shape of a rocket, the height of which is
proportional to the concentration of antigen in the well. One limitation
of this technique is the need for negatively charged antigens. Some
proteins such as immunoglobulin are not sufficiently charged to be
quantitated by RIE, nor it is possible to quantitative several antigens in
a mixture at the same time.
Materials and Equipment
2% agar in barbitone buffer
Human serum albumin (HAS)
Anti- HAS
Phosphate – buffered saline (PBS)
Agarose preocated slides
Leveling table
Gel punch
water bath held at 56oC
Electrophoresis tank and power pack
Method
1. Melt agar in a microwave oven or on hot plate and allow cooling
to 56oC.
2. Add antiserum to test tube and dilute for use (as a guide, use
0.1 ml of antiserum and add 0.9 ml PBS).
3. Mix and warm to 56oC
4. Add 2 ml agar to the diluted antiserum and mix.
5. Pour onto precoated slides on the leveling table.
6. Punch wells.
7. Fill the wells with antigen solutions – in the range 50-500 µg /
ml should be suitable.
8. Run the electrophoresis at about 8 mA / slide, 5- 10 V/ cm.
9. Run for at least 2 h.
10.
The peaks may be measured immediately, but this is
easier after staining
11.
Plot the height of the peaks against the antigen
concentration if a standard curve has been determined.
17
If the slide was run until the precipitin arc become stationary, the
relationship of peak height to antigen concentration is linear.
Technical note
The assay can not be used directly to quantify Ig G as both the antigen
and antibody would be moving in the same direction in the
elecrophoretic field. However, the electrophoretic mobility of the Ig G
antigen may be altered by carbamylaltion.
Photograph showing the
immunoelectrophoresis
development
of
Rockets
after
Observation
Observe the agar gel after electrophoresis against dark background.
Measure the height of each peak and plot it against the antigen
concentration loaded in the well and determine the concentration of
unknown antigen.
Result
The
concentration
of
unknown
antigen
from
immunoelectrophoresis was found to be ----------------------
18
Rocket
Experiment No. 6
Study of immunoprecipitation
Aim: Quantitative Precipitation Test
Principle
This technique is developed by Heidelberger and Kendall. It is the
basis of all quantitative studies of antigen-antibody interaction.. In this
technique increasing amount of antigen is added to a constant amount
of antibody in test tubes and the weight of precipitate formed in each
tube is determined. One important observation is that the precipitation
obtained with divalent F(ab’)2 fragment of antibody is less efficient
than with antibodies containing intact Fc region. Thus Fc region of
antibody selected should be intact.
Materials
Antiserum, e.g. anti-human serum albumin (aged or inactivated by
heating at 56OC for 15 min)
Human serum albumin (HAS) (1mg/ml)
Phosphate-buffered saline (PBS)
0.1M Sodium hydroxide
Method
1 Add antigen, PBS and antiserum to a series of numbered tubes
(suitable for centrifugation) according to the protocol. ( for antisera
with high or low antibody content it will be necessary to increase or
decrease the range of antigen concentrations used.)
Protocol
Reagent
1
2
3
4
5
6
7
8
9
10
Antigen
0
10
20
50
100 150 200 250 350 450
(µl)
PBS (µl)
450 440 430 400 350 300 250 200 100 0
Antiseum 100
(µl)
100
100
100
100
100
100
100
100
100
2 Mix the reactants thoroughly.
3 Incubate at 37oC for 1 h and then at 4oC overnight ( for accurate
determinations, these incubations can be extended for up to 10 days
at 4oC. However for demonstration purpose using high-titred and highavidity antisera, illustrative curves may be obtained with only 30 min
incubation at 37oC and 30 min at 4oC.)
19
4. Centrifuge at 3000 g for 5 min at 4oC and remove supernatant. An
angle-head rotor should be used as the precipitate is then formed at
the side of the tube, thus facilitating removal of the supernatant.
5. Check each supernatant for free antigen antibody using a sensitive
technique, e.g. single radial immunodiffusion
6. Wash the precipitate twice by centrifugation with cold PBS.
7. Redissolve the final precipitate in 0.1 M sodium hydroxide ( the
volume to be used depends on the spectrophotometer cuvettes
available, but should be about 1ml for the amounts of reagent used
here).
8 Measure the absorbance at 280 nm and plot a graph of the
absorbance units of the redissolved precipitate against the amount of
antigen added.
Observation and Calculation
1 Determine the antibody content per ml of antiserum.
If the supernatant from each tube is examined for the presence of
excess antibody or antigen, there will be one point at which no free
antibody or antigen can be detected. This is the point of equivalence,
which occurs just before maximum precipitation. The amount of
precipitate increases after the equivalence point because of continued
incorporation of antigen into the complex. Eventually soluble
complexes are formed in the antigen excess and the amount of
precipitate decreases.
Graph
20
In the graph, at equivalence, the precipitate contains x µg of total
protein; if this includes y µg of antigen then there is ( x-y) µg of
antibody. This is the total amount of antibody in the volume of serum
used.
Result
The total amount of antibody in the serum by quantitative
immunoprecipitation technique was found to be -------------------------
21
Experiment No. 7
Precipitation in gel
Principle
If antigen and antibody are placed in two adjacent wells in agar, they
will diffuse into the agar and set up two opposing concentration
gradients between the wells; at a point of optimal proportions between
these gradients a line of precipitation will form. Thus, the analysis will
be carried out with the reactants at their equivalence point, without
the need for empirical determination.
Materials and Equipment
Barbitone buffer
Agar
Method
Buffered Agar
1 Mix 2 g of agar with 50 ml of distilled water and dissolve by heating
in a microwave oven.
2 add 50 ml of hot barbitone buffer and mix well. The agar may be
stored at 4oC for many weeks
Note: The agar must be bought specifically for electrophoresis;many of
the culture agars are not suitable for this purpose
Precoating Glass Slide
Gel precipitation techniques may be conveniently performed on glass
microscope slides and the gel then dried down onto the glass for
permanent storage. To ensure the good adhesion of the gel to the
slide it is necessary first to coat the slide with a thin layer of agar
which is allowed to dry, before layering on the analytical agar gel.
Material and Equipment
Glass microscope slides
Agar
Method
1 Dissolve 0.5 g of agar in 100 ml of distilled water as above.
2 Pipette the agar solution onto clean, dry slides. Add enough to cover
sparingly one surface of the slide.
3 Dry the slide and store at room temperature until required.
22
Experiment No. 7 continuation
Aim: Single Radial Immunodiffusion (SRID)
Principle
In this technique as the antigen diffuses radially a ring of precipitation
forms around the well and moves outwards, eventually becoming
stationary at equivalence. At equivalence, the diameter and area of
the ring are related to the antigen concentration in the well. Using
standard antigen concentration calibration curve may be constructed
to determine unknown concentrations of the same antigen.
The optimal concentration for the antiserum will, of course,
depend upon the strength of the antiserum and antigen as the
diameter of the precipitation ring is inversely proportional to the
antiserum concentration. In practice, with rabbit antisera to human
Ig G, a final dilution of approximately 1: 40 in the agar is suitable for
measuring Ig G concentration in the range of 50- 200 µg/ml. However,
this is only guide; a standard curve should be determined for each
antiserum.
Materials and Equipment
2% agar in barbitone buffer
Precoated slides
Antiserum, e.g. anti-human Ig G
Standard antigen solution, e.g. human Ig G
Phosphate- buffered saline (PBS)
Flat, level surface (use a spirit level)
Gel punch
Humid chamber ( plastic box with damp filter paper)
Pasteur pipettes
Water vacuum pump
Method
1. Melt the agar in a microwave oven and transfer to a 56oC water
bath. This temperature will keep the agar molten but is low enough to
avoid denaturation of the antibody.
2. Dilute the antiserum with PBS. Typically, add 75 µl of an antiserum
to 1.9 ml of PBS and warm to 56oC.
3. Add the diluted serum (~ 2ml) from step 2 to 1ml of agar at 56oC
and mix well.
4. Layer the agar onto a precoated slide standing on a leveled surface
and allow to set.
23
5. After the agar has set, use a gel punch to cut about eight wells per
slide. The well should be 2-3 mm in diameter, and must have vertical
sides.
6. Remove the agar plug with a Pasteur pipette attached to a water
vacuum pump.
7. Fill each of four wells with standard solutions of 50, 100, 150 and
200 µg/ml of Ig G. Use the other wells for the Ig G solutions of
unknown concentrations. Maintain a standard volume by filling the
wells quickly until the meniscus just disappears. Alternatively a
measured volume, such as 10 µl, may be accurately pipetted into each
well.
8. Leave the slide in a humid box to equilibrate. (Although a
satisfactory standard curve may be obtained by overnight
equilibration, the point will better approximate to a straight line if the
slide is allowed to equilibrate longer: Ig G and Ig A for 48 h, Ig M for
72 h. Ig G concentrations may also be determined by incubation at
37oC for 4 h.)
Measurement of the precipitation rings
The diameter of each ring may be measured either directly using a
magnifying glass with a µm scale or, after staining, with a plastic ruler.
Direct Measurement
Hold the slide over a black background and illuminate it from the side.
Measure the rings from the reverse side through the glass plate; do
not rest the magnifying glass on the gel. If the rings are not distinct,
soak the slides in 4% tannic acid for 1 min to increase resolution. (This
is not a permanent preparation)
24
Photograph showing precipitation rings of variable diameter
formed because of different concentrations of Ig G.
Stained Preparations
1 Wash the slide for 24 h in several changes of PBS to remove free
protein from the agar.
2 Cover the slide with good-quality, lint-free, filter paper and dry
overnight.
3 Remove the filter paper after dampening it slightly.
The slide may then be stained with any protein dye, but Coomassie
blue can be used.
Mixture for staining solution
Coomassie brilliant blue R- 250 (1.25g)
Glacial acetic acid ( 50ml)
Distilled water (185 ml)
Method
1 Dissolve the Coomassie dye in the glacial acetic acid and distilled
water.
2 Stain the slide for 5 min and differentiate in the same solution
without the dye. Staining
with this dye is reversible so do no leave the slide too long in the
destaining solution.
3 Place the dry, stained slides in a photographic enlarger and measure
the diameter of the precipitation rings with a ruler.
The staining solution may be stored for several weeks in a stoppered
bottle. The destaining solution can be regenerated by passing through
powdered charcoal.
Observation and Calculation
Observe the slide and Measure the diameters of the rings and plot
against the antigen concentration. A calibration curve is constructed
from the diameter of the precipitation rings formed at equilibrium b Ig
G standards of known concentration. The concentrations of unknown
samples can then be determined with reference to the standard curve.
Result
The concentration of unknown from single radial immunodiffusion
technique has been found to be ------------------------
25
Experiment No. 8
Aim: Double diffusion assay
Double diffusion in two dimensions
Principle
In this technique the antigen and antibody are allowed to migrate
towards each other in a gel and a line of precipitation is formed where
the two reactants meet. As this precipitate is soluble in excess antigen,
a sharp line is produced at equivalence, its relative position being
determined by the concentration of the antigen and antibody in the
agar. The local concentration of each reactant depends on : (a)
absolute concentration in the well: (b) its molecular size; and (c) the
rate at which it is able to diffuse through the gel. Multiple lines of
precipitation will be present if the antigen and antibody contain several
molecular species.
The particular advantage of the technique is that several
antigens or antisera can be compared around a single well of antibody
or antingen.
Materials and Equipment
2% agar in barbitone buffer
Antigen and antibody solutions
Gel punch
Pasteur pipettes
Water vacuum pump
Antigen concentration: initially use 1 mg/ml, but vary the
concentration to obtain optimal results
Antiserum: use whole anti-Ig G, non-absorbed. Specific anti-Ig G
sera available commercially are often absorbed with light chains to
render them class specific.
Method
1 Melt the agar in a microwave oven or on hot plate.
2 Pour agar on a precoated slides; use a leveled surface.
3 Punch pattern required.
4 Such out agar plugs with a Pasteur pipette connected to a water
vacuum pump.
5 Fill the wells with antibody or antigen until the meniscus just
disappears.
6 Place the slide in a humid chamber and incubate overnight at a
constant temperature.
26
Technical note : the rate of diffusion is temperature dependent.
Precipitin lines can often be seen within 3 h at 37oC
Antigen and antibody pattern
(A)
(B)
Interpretation of Results
The figure A displays
(a) Reaction of identity: This occurs between identical antigenic
determinants; the lines of precipitation fuse to give on continuous arc.
(b) Reaction of nonidentity: Where two antigen do not contain any
common antigenic determinants the two lines are formed
independently and cross without any interaction.
(c) Reaction of partial identity. This has two components: ( I)
those antigenic determinants which are common to both antigens give
a continuous line of identity; (ii) the unique determinats(s)
recongnized on one of the antigens give(s), in addition, a line of nonidentity so that a spur is formed. Of course, the antiserum may
recognize unique determinants in both antigens; this would give rise to
two spurs;
27
All these concepts of identity and non-identity are in terms of
recognition by the antiserum. An antiserum recognizing many
determinants on the antigen molecules is necessary for the
demonstration of all these features
Result ------------------------------------------------------------------
28
Experiment No. 9
Aim: To perform Enzyme Linked Immunosorbent Assay (ELISA)
Principle
It is an extremely scientific technique which dose not require any
precautions as against radioimmunoassay.
It is more stable than labeled (radioactive) iodinated proteins. It
is easy to handle, simple and multiple samples can be analyzed easily.
The assay for an antigen depends on being able to couple highly
specific antibody to enzyme and a solid substrate such as plastic beads
or plates (the inner wall of the wells where assay is done).
The antigen is immobilized on a solid surface. The antibody
specific to this antigen is added to the plate which binds to the
antigen. Then an antibody conjugation is done with the antigen again.
This binds to antigen portion of the first or primary antibody. This
complex can be detected by enzyme substrate reaction. The intensity
of this chromogenic reaction is measured.
Generally the polystyrene plates are employed whose binding
capacity is 300 ng/cm. The binding of protein with polystyrene is not
constant-may be electrostatic as polystyrene has positive charges.
The various modifications of the basic ELISA technique are as follows:
1. Direct ELISA
Here the first antibody is conjugated with an enzyme, which binds
specifically to the antigen. This complex is then assayed with the help
of a chromogenic substrate reaction.
2. Indirect ELISA
The antibody conjugated with enzyme is directed against another
antibody, which specifically binds to the antigen in use. This is in
common use as it avoids unnecessary wastage of the primary
antibody-enzyme conjugate.
3. Sandwich ELISA
The plate is first coated with the antibody, which binds to the plate
through its Fc receptors. Then antigen is added. The antigenic receptor
present on antibody binds to the antigen added. The second antibody
conjugated with enzyme is added after the addition of another
antibody against antigen, but of different origin which also specifically
binds to the same antigen. The complex is then assayed using
chromogenic reaction.
29
4. Competitive ELISA
In this technique the antibody is first incubated in solution with the
sample containing antigen. The antigen and antibody mixture is then
added to an antigen coated microtitre plate. Addition of an enzyme
conjugated secondary antibody can be used to quantitate the amount
of primary antibody.
Enzyme and substrate available for ELISA
A number of enzyme have been employed for ELISA including
alkaline phospatase, horse radish peroxidase and p-nitrophenyl
phosphatase.
Enzyme
Phosphatase
in carbonate buffer
Substrate
p-Nitrophenyl phosphate 1mg/ml
containing 0.5 mM magnesium
chloride.
Peroxidase
mg in 12.5 ml of
Ortho phenylene diamine (OPD) 5
carbonate buffer, add 5 µl of 30
% hydrogen peroxide.
Method
1. Coat the polystyrene ELISA plate with the protein ( e.g. BSA) i.
e. antigen, 100 ul in a sensitizing solution carbonate –
bicarbonate buffer (pH 9) at an optimal concentration.
2. Incubate for 2 hrs at 37oC and transfer to 4oC overnight.
3. Next day wash the plate twice with PBS.
4. Fill the plate with 200 µl of blocking reagent (3% skimmed milk
in PBS) to block the exposed area which does not have antigen
bound to it.
5. Add different dilutions of serum - the primary antibody ( 100 µl
) to each well.
6. Incubate for 1 hr at 37 oC.
7. Wash twice with PBS + 0.05% Tween 20.
8. Add 100 µl l of second antibody conjugated with horse radish
peroxidase (diluted 1:2000) to each well.
9. Incubate for 1h at 37 oC
10.
Wash twice with PBS + Tween.
11.
Add substrate solution (H2O2 + OPD) to each well.
12.
Stop the reaction by adding 2 N H2SO4 after 15 to 20 min.
13.
Check O.D. of each well at 492 nm.
30
14.
Plot a graph: O. D. vs. concentration of primary antibody
on a semi-log graph paper.
15.
Leave the first two columns of the plate as blank. To this
add everything except primary antibody. This will act as ‘zero
intensity’ well while taking the O.D.
Photograph
of
ELISA
plates
showing
development of different
colors
of
variable
intensity. Development of
color depends upon the
chromogenic
substrate
and enzyme used during
ELISA
31
Experiment No. 10
Aim: To perform western blot analysis
Principle
Identification of specific protein in a complex mixture of protein
or antibody to a given protein can be accomplished by western blot. In
this technique protein is electrophoretically separated on a
polyacrylamide slab gel. The protein bands are transferred on a
nitrocellulose membrane by electrophoresis or by diffusion such that
the membrane gets a replica of the gel containing protein bands. The
individual protein bands are identified by flooding the membrane with
primary antibody. After this the antigen – antibody can be detected by
adding a secondary antibody conjugated with enzyme. The band is
visualized by adding substrate which being a chromogen on conversion
to product gives color. The intensity of color shows the quantity of
antigen – antibody complex formed.
Western blotting
(also known as protein blotting or
immunobloting) is a rapid and sensitive assay for detection and
characterization of proteins. Western blotting technique exploits the
inherent specificity of antigen-anitbody interaction to identify specific
antigens by polyclonal or monoclonal antibodies.
SDS-PAGE:
Sodium Dodecyl Sulphate –Polyacrylamide Gel Electrophoresis
(SDS-PAGE) is carried out in a discontinuous buffer system wherein
the reservoir buffer is of different pH and ionic strength from the
buffer used to cast the gel. The SDS-polypeptide complexes in the
sample applied to the gel are swept along by a moving boundary
created when an electric current is passed between the electrodes.
After migrating through the stacking gel of high porosity, complexes
get deposited in a very thin zone on the surface of the resolving gel.
On further electrophoresis, polypeptides get resolved based on their
molecular size in the resolving gel.
Western Blotting
Blotting is transfer of resolved proteins from the gel on to a surface of
a suitable membrane, done commonly by electrophoresis and referred
to as electro blotting. The gel is place in contact with nitrocellulose
membrane which is then sandwiched between filter paper, two porous
pads and two plastic supports. The entire set up is then placed in an
electrophoretic tank containing blotting buffer. The protein gets
transferred to the corresponding position on the membrane as
resolved on the polyacrylamide gel, forming the mirror image of the
32
gel. Protein of interest on the membrane is further located by
immunodetection.
Immunodetection
The transferred proteins bound to the surface of nitrocellulose
membrane are detected using immunological reagents. This process is
known as immunodetection. All the unoccupied sites on the
membrane are first blocked with an inert protein, a detergent or any
other suitable blocking agent. The membrane is then probed with a
primary antibody specific to the protein of interest. The Ag-Ab complex
formed on the membrane is then identified using an enzyme – labeled
secondary antibody and a suitable substrate on the enzyme, which
results in a coupled band on a nitrocellulose membrane, referred to as
blot development
Methodology for SDA-PAGE
1. Assemble the plates for casting the gel
2. Clamp the assembly of plates to fix it in a gel casting apparatus.
Ensure the assembly is leak proof by filling water between the
plates. Use slilicon grease for making water-tight seal around the
spacer.
3. Add 50 µl of APS solution to 5 ml of SDS separating gel mix and
pour the gel solution between the plates till the level is 2 cm
below the top edge of notched plate.
4. Add 200 to 250 µl of water to make the surface even.
5. After the gel is set ( approximately 20-30 min.), wash the top of
the separating gel with distilled water and drain off the water
completely.
6. Add 20 µl of APS solution to 2 ml of stacking gel mix and pour
directly onto the polymerized separating gel
7. Insert the comb into the gel solution carefully without trapping
any bubbles, about 1 cm above the separating gel. The stacking
gel will set in approximately 10 min.
8. Add 25 µl of sample loading buffer to protein sample.
9. Add 25 µl of sample loading buffer to 25 ul of protein marker
10.
Place it in a boiling water bath for 5 min.
11.
After the gel has set, carefully remove the comb and the
bottom spacer. Wash the wells immediately with distilled water
to remove non-polymerized acrylamide. Fill the bottom reservoir
with reservoir buffer and carefully fix the plate to the apparatus
without trapping any ail bubble between the buffer and the
bottom of the gel. Fix the plates to PAGE apparatus. Fill the top
reservoir with reservoir buffer.
33
12.
Load 30 µl protein marker in well 1, 40 µl of protein
sample in well to 2 and 5 µl of protein sample in well 4. Note
down the order of loading. Connect the cords to the power
supply according to the convention red: anode, black: cathode
13.
Set voltage to 100 V and switch on the power supply.
14.
When the dye front comes to 0.5 cm above the bottom of
the gel, turn off the power. This will take approximately 1 to 1½
hrs.
15.
Remove the gel plates and gently dry the plates apart
using a spatula or similar tool, not at the notch.
16.
Transfer the gel to a tray containing water, wash the gel
for 1 to 2 min at room temperature.
17.
Decant water, cut the lane along lane 3.
18.
Transfer the lane 4 i.e. protein sample in 10 ml of blotting
buffer in a petri dish. Keep at room temperature for 10 min.
Following incubation proceed for electroblotting.
19.
To the gel piece ( lanes 1 and 2) add minimum of 20 ml
water.
20.
Decant the water, add minimum 20 ml of Ezee Blue Stain.
Stain at room temperature fro 1-2 hr.
21.
For uniform staining and washing, place the tray on a
rocker or shake intermittently 10 to 15 minutes.
22.
Decant the staining solution add minimum quantity of
water to cover the gel.
23.
Cover the tray and leave it overnight at room temperature.
Enzyme and substrate for western blot analysis
A number of enzymes have been employed for western blot developing
– alkaline phosophatase and horse radish peroxidase.
Alkaline phosphatase
Solution A
NBT – Nitroblue tetrazolium
3 mg in 0.25 ml buffer containing 0.1M Tris. HCl pH 8.8, 0.1M NaCl,
0.005 M MgCl2.
Solution B
BCIP- 5 – bromo – 4 – chloro-indolyl phosphate 1.5 mg in 10 µl
dimethyl sulphoxide, add 0.25 ml of 2 M Tris. HCl pH 9.8
Mix A and B and makeup to 10 ml.
Horse radish peroxidase
A. 30 mg of 4 – chloro – 1- napthol in 10 ml methanol
34
B. 30 µl of H2O2 in 40 ml TBS
Mix A and B at room temperature.
Transfer of protein by diffusion
Transfer of protein bands from an acrylamide gel onto a membrane, by
diffusion by the capillary action of buffer is capillary blotting.
Solutions to be prepared
1. Urea buffer
Sodium chloride : 0.29g
EDTA
: 0.07g
Tris
: 0.12 g
Urea
: 24.00g
Adjust the pH to 7.0 and make up the volume to 100ml with distilled
water.
2. Transfer buffer.
Sodium chloride
: 2.9 g
EDTA
: 0.7 g
Tris
: 1.2 g
Adjust the pH to 7.0 and make up the volume to one litre with distilled
water.
3. Phosphate buffered saline (PBS)
Sodium chloride
Potassium chloride
Disodium hydrogen phosphate
Potassium dihydrogen phosphate
Distilled water: 1 litre
: pH 7.2
: 8.00 g
: 0.20 g
: 1.15 g
: 0.20 g
Method
1. Remove SDS from the gel by washing the gel with urea buffer
for 30 min
2. Measure the size of the gel and cut Whatman No. 3 paper 6
sheets and nitrocellulose membrane 2 sheets.
3. Assemble the sandwich as given in the figure
4. Put rubber bands to keep the sandwich intact.
5. Immerse the whole sandwich in transfer buffer
6. Keep a lightweight on top to prevent the floating of the sandwich
and to make sure that the sandwich is kept immersed
completely. Avoiding trapping any air bubble while preparing the
sandwich assembly.
7. Keep for transfer for 12 to 18 hrs.
35
8. Remove nitrocellulose membrane and proceed for antibody
labeling or can be stored in a box for more than 6 months.
Electrophoretic Transfer or Electroblotting
1. Soak the gel, Whatman paper and nitrocellulose in transfer
buffer (80% running buffer + 20 % methanol.)
2. Assemble the blotting sandwich within the blotting cassette
3. Take care to avoid the air bubbles between the gel and NC
membrane.
4. Insert the cassette into the apparatus filled with blotting buffer
and connect blotting unit to power supply as per the convention.
Red: anode, black: cathode
5. Electrophores the sample at 50 V for 2 hr for blotting to occur.
6. Remove the NC membrane gently from the cassette and place
the membrane in 10 ml of freshly prepared blocking buffer taken
in a petri dish. Leave it overnight at 4oC.
Procedure for developing western blot
1. Take the NC paper and shake in the blocking agent for 1-2 hours
( 3% milk in TBS, 10 mM tris – HCL pH 7.3 containing 0.15 M
sodium chloride).
2. Transfer the paper to 1 % primary antibody solution made in
blocking agent. Keep at room temperature for 1 h with constant
shaking.
3. Rinse the paper in distilled water and keep in another container
with TBS for 10 min at room temperature.
4. Repeat step 3 with fresh TBS.
5. Add secondary antibody conjugated with alkaline phosophatase (
1:2000 dilution) in 1 % milk in TBS.
6. Shake for 1 hr at room temperature.
7. Rinse in distilled water and wash in TBS for 10 min twice.
8. Add the substrate solution (NBT + BCIP) in dark
9. Color develops within 10 min. Take a photograph immediately.
36
Generalized diagram of western blot
Observation
On staining SDS-Polyacrylamide gel, different proteins will appear as
dark blue bands against a light blue background. On immunodetection,
a single blue band will be observed on NC membrane.
The bands obtained on the polyacrylamide gel and after
immunodetection on NC membrane are compared and band of interest
is located. The relative position of the band on NC is matched with the
molecular weight marker and its molecular weight is determined.
Note: Here students are expected to write their own observation by
taking the reference of above sentences.
Result
1. The band on the Nitrocellulose membrane indicates the ---------protein detected by antibody (------------------).
2. The molecular weight of the detected protein has been found to
be ---------
37
Experiment No. 11
Aim: Purification of Immunoglobulin
Principle
Proteins are charged molecules and are associated with solvent
molecules in solution. When this association is disturbed by addition of
inorganic salts like ammonium sulphate, molecules tend to aggregate
and precipitate out of the solution as this neutral salt affects the
balance between electrostatic forces tending to keep the protein in
solution and hydrophobic forces that cause proteins to aggregate and
precipitate in aqueous solution.
Ammonium sulphate is non-toxic and stabilizes protein in solution.
Method
I. Ammonium sulphate fractionation
1. Take serum in a conical flask.
2. Add 100% ammonium sulphate drop wise with constant mixing
to 33.3% saturation in ice.
3. Keep it in ice for 20 min.
4. Centrifuge it at 5000 rpm for 5 min.
5. Suspend the pellet in PBS and dialyse over night against
phosphate buffer
II. Pre treatment of Dialysis Bag
1. Boil the dialysis bag in the distilled water for 10 min.
2. Boil it for 30 min. in 0.2 M NaHCO3.
3. Wash this bag with distilled water.
4. Boil it in 1 mM EDTA solution.
5. Wash the bag in distilled water and store in 50% alcohol.
III. Affinity chromatography
1. Suspend affinity chromatography material ( Cyanogen activated
sepharose 4B) in phoshate buffer.
2. Wash 5 times with 1mM HCl
3. Wash with coupling buffer ( 0.1 M NaHCO3 pH 8.3 and 0.5 NaCl)
to remove HCl.
4. Dissolve the protein ( anti -Ig G) to be couple in coupling buffer
and take O.D. at 260 and 280 nm.
5. Mix the protein and the above treated gel (sepharose 4 B) and
keep for 2 h at room temperature.
6. Transfer these then to blocking reagent that is 0.02M glycine pH
8 and keep for 2 h.
7. Wash it with coupling buffer and store it overnight at 4oC
38
8. Pack the above beads into the column and layer the Ig G fraction
in PBS on the top of the column.
9. wash the column twice with PBS.
10.
Elute with 0.02 M Glycine pH 8 and thrice with acetate
buffer (0.1 M sodium acetate)
11.
Elute with acetate buffer pH 6.0 to obtain Ig G1, pH 4.5 to
obtain Ig G2a and pH 3.5 to obtain Ig G 2b.
12.
Take O. D. at 280 and 260 of all the fractions and
calculate the total amount of Ig G Purified.
(1.55 X O. D. 280 – 0.76 X O. D. 260 = mg protein/ml).
Photograph showing Cynogen activated sepharose 4 B
column used for immunoglobulin purification
Observation
Collect the various fractions of filtrate and measure its ODs and
determine the concentration of Ig G in the fractions.
Result
The amount of immunoglobulin in the purified fraction has been
found to be -----------------
39
Experiment No. 12
Aim: Preparation of lymphocyte
Principle
The separation of RBC and the immunological cells i.e. lymphocytes is
based on the differences in density with reference to gradient. In ficol
gradient RBCs settle down and the lymphocytes are found in the
interphase. This forms the basis of separation of lymphocytes from the
human blood samples.
Material
Human blood
Alsever’s solution
Ficol
Haemocytometer
Trypan blue
Method
1. Collect 10 ml of venous blood in 2 ml of Alsever’s solution ( 2
ml).
2. Take 3 ml of blood and add it to a beaker containing Alsever’s
solution.
3. Measure volume and add PBS in a tube and make up volume to
10 ml and mix well.
4. Take 4 ml of ficol in another graduated tube.
5. Add the blood slowly and carefully onto this, so that it forms a
red layer over the transparent layer of ficol, till total volume is
12 ml.
6. Centrifuge at 750 rpm for 25 min.
7. Four layers will form : plasma (on top), peripheral blood
mononuclear cells (PBMC), ficoll and pellet of RBCs.
8. Remove PBMC layer.
9. Add PBS and centrifuge at 1500 rpm for 10 min. Repeat thrice.
10.
Add to 50 µl of above preparation, 50 µl of 0.4% Trypan
blue.
11.
Count the viable cells after loading the sample on a
haemocytometer. Dead cells pickup the dye.
Other Method
Materials
Human blood
40
Endotoxin-free heparin
Hank’s balanced salt solution ( sigma)
LymphoprepTM (Nycomed Amersham)
Pasteur pipettes
20 ml centrifuge tubes
RPMI 1640 containing 2 mM L-glutamine, 1mM sodium pyruvate,
100U/ml penicillin, 100 µg/ml streptomycin, 0.5 µg/ml fungizone and
10% heat-inactivated pooled human serum ( e. g. human AB serum or
autologous serum
Method
1. Collect human peripheral blood into tube containing endotoxinfree heparin (10 U/ml blood)
2. Add an equal volume of Hank’s balanced salt solution and mix
gently by inversion
3. Overlay 10 ml aliquots of the diluted heparinized blood onto 5 ml
of LymphoprepTM. Then centrifuge at 800 g for 20 min at 20oC
4. Then middle interface contains the lymphocytes. Remove cells
using the sterile Pasteur pipette, taking care not to disturb the
upper layer.
5. Wash the cells with 10 ml of Hank’s balance salt solution. Pellet
the cells by centrifugation at 250 g for 15 min at 20oC
6.
Resuspend the cells for culturing in RPMI 1640 containing 2
mM L-glutamine,
1mM sodium pyruvate, 100U/ml penicillin, 100 µg/ml
streptomycin, 0.5µg/ml
fungizone and 10% heat-inactivated pooled human serum ( e.
g. human AB serum
or autologous serum.
Observation
Observe the isolated lymphocyte under microscope and calculate its
number and report.
Result
Lymphocyte isolation has been performed successfully and its number
in the given serum sample has been found to be ------------------------
41
Experiment No. 13
Aim: Agglutination reaction with reference to blood grouping
Introduction
It was Karl Landsteiner who in 1900 grouped human blood into
four groups based on the presence of two antigens on the surface of
the blood cells. These groups are named as A, B, AB and O. The blood
typing and blood group identification is performed by antisera
containing high titers of anti-A and anti-B antibodies.
Requirements
Blood sample
Saline solution
Glass vial
Cotton
Disposable needle
Slide
Antiserum A
Antiserum B
Tooth pick
Method
1) Collect blood sample in 1 ml saline solution in a small vial by
scrubbing the middle finger with a piece of cotton saturated with
70% alcohol and pierce it with a disposable sterile needle.
2) Use only 2 or 3 drops of blood to mix with saline.
3) Place a drop of this blood suspension on the two places on the
slide and add a drop of antiserum-A to the right drop and antiserum
– B on to the left.
4) After mixing each slide with separate toothpicks, examine the
spots for agglutination for 2-3 minutes and record the results.
Observation
If the agglutination occurs in the suspension to which anti-A is
added, the group is A, if it occurs in B the group is B. If
agglutination occurs in the suspension of both antiserum-A and
antiserum-B, the blood group is AB, and if there is no agglutination
reaction or absence of agglutination in any of the suspension, the
blood group is O.
Result
The blood group of the person under investigation has been found to
be ---------42
Experiment No. 14
Aim : Blood examination for Rh factor
Principle
In 1940, Landsteiner and Wiener found
that rabbit sera
containing antibodies against red blood cells of the rhesus monkey
would agglutinate the red blood cells of some human beings. The
antigen was first designated as Rh factor. Later it was found to exist in
the form of six antigens: C, c, D, d, E and e. Among these six
antigens, D is responsible for the Rh positive condition.
Identification of Rh factor is performed by a similar technique for
identification of bood groups. The only difference is that the whole
blood is used and test is carried out at
higher temperature (45oC). Because the antibodies in typing sera are
of incomplete albumin variety, which will not agglutinate human red
cells when diluted with saline.
Requirements
Blood sample
Slide warming box
Antiserum-A
Antiserum-B
Antiserum-D
Toothpicks
Ethanol
Cotton
Method
1) Use a small amount of blood, and agitate properly. The
agglutination appears in the form of fine clumps.
2) To get the proper reaction, use only a drop of blood of less than 3
mm diameter.
3) Add a drop of antiserum –D to the blood on a slide overlying in the
warm box.
4) Mix it with tooth pick and watch exactly after two minutes.
5) Agitate mixture on the slide by slowly rotating the box.
6) At the end of 2 minutes, examine the agglutination.
Result
If agglutination does not occur, blood is Rh negative.
Note: Kits for bood group idetification are commercially available and
are efficient, rapid. These Kits can be used for this practical.
43
Experiment No. 15
Aim: Widal test using tube agglutination reaction
Principle
The tube test is performed for quantifying agglutinating antibodies in
the serum of a patient suffering from typhoid. Add suspension of dead
typhoid bacterial cells to a series of tubes containing the diluted
patient serum. This technique confirms the actual causal organism of
typhoid. This test is based on the principle if higher the titer greater is
the response of the reaction product. Successive daily test on a patient
serum reveal no or low antibody titer from day to day , the organism is
not Salmonella. It is possible only in case when titer increases daily.
Requirements
Sample of blood serum
Serological tubes
Physiological saline (0.85% NaCl)
Pipette
Incubator
Glass marker.
Method
1) Take a blood sample from patient suffering from typhoid in a
serological tube and collect few more tubes for making the sample.
2) Add 0.5 ml of saline to each tube of blood serum sample (1: 10)
as well as other tubes
3) Transfer 0.5 ml blood serum from tube 1 to next tube, mix it and
transfer 0.5 ml from this tube to next, mix it and again transfer into
the next tube subsequently. The last tube should not have serum
and will contain only saline and can be used as control.
4) Transfer 0.5 ml of S. typhimurium antigen in each tube with the
help of pipette, mix the antigen thoroughly and keep on a water
bath at 37oC for 30 min.
5) Examine each tube for agglutination after centrifugation and
record the titre in which agglutination has occurred.
Result
Agglutination will show that patient is suffering from typhoid.
44
Experiment No. 16
Aim: Isolation of DNA from Blood Cells
Principle
DNA is the macromolecule of paramount importance since it is the
store house of entire information of all the processes of the body. DNA
can be isolated from any nucleated tissue cells. Blood is the most
widely available tissue with nucleated leucocytes and hence is used for
obtaining DNA.
Several methods are available for deproteinizing cell digest
during isolation of DNA. These include the classical phenol Chloroform
extraction, extensive dialysis, use of filters, and precipitation with
saturated sodium acetate solution. Several commercial methods are
available which use column, beads etc.
Leucocytes suspended in hypotonic solution like distilled water,
non-ionic detergent like NP-40, swells and lyse. NP40 breaks cell
membrane of leucocytes and release nuclei. Nucei can be collected as
a pellet after centrifugation. Lysed RBCs and other cell debris are
removed as supernatant . Nuclear pellet is then suspended in a lysis
buffer containing a strong detergent like SDS and a highly active
protease – Proteinase K.
SDS and Proteinase K degrade the protein proteins; chloroform
denatures and extracts protein and lipid. DNA present in the aqueous
phase is precipitated by ethanol and isopropanol.
Requirement
Blood sample
Microcentrifuge tubes 1.5 ml
Micropipettes
UV Transilluminator or Gel documentation system
Electrohoresis unit
Reagents
1. Solution I (10X)
Tris buffer
16.05 gm
KCl
37.28 gm
MgCl
2 10.16gm
Dissolve in 100 ml distilled water and adjust the pH 7.8
45
2. Lysis Buffer
Lysis buffer is used fresh each time
Distilled water 71 ml
Solution I
10 ml
8M NaCl
10 ml
20% SDS
5 ml
0.7 M EDTA
4 ml
3) Tri – EDTA buffer (TE Buffer)
10 mM Tris buffer and 1 mM EDTA, pH 8.0
Filter through 0.22 u micro-filter and store at room temperature.
4) Chloroform
5) Absolute ethanol or isopropanol.
Note: All the solution should be sterilized by 0.22 µ micro-filter
assembly before use and stored at room temperature.
Method
1. Take 1 ml of anticoagulated blood (EDTA, citrate and heparin) in
a 1.5 ml of microcentrifuge tube
2. Centrifuge at 10000 rpm for 2-4 min and discard plasma without
disturbing cell pellet
3. Add 1 ml of distilled water and 40 ul of NP40 to the cell pellet
and mix well on end to end mixer for 5 min so as to lyse RBCs.
4. Centrifuge at 10000 rpm for 2 min to pellet leucocytes nuclei.
5. Add 1 ml of cell washing buffer to wash the nuclear pellet.
6. Mix well and spin at 10000 rpm for 1-2 min. Discard the
supernatant.
7. Add 500 µl of lysis buffer. Mix well so as to disturb the nuclei
pellet and have even suspension of nuclei.
8. Add 10 µl proteinase K (100 gm / ml)
9. Mix well and incubate at 54-58oC for 30-60 min. check whether
the solution is clear, if any suspending particles are seen,
incubate till solution becomes clear. Shake every 10 min.
10.
After incubation cool the solution.
11.
Add 200 µl of 5 M sodium acetate solution and mix well for
1-2 min.
12.
Add 500 µl of chloroform. Mix well on end to end mixer for
5-10 min.
13.
Centrifuge at 10000 rpm for 3-5 minutes.
46
14.
Transfer the upper aqueous phase carefully to a clear 1.5
ml micro-centrifuge tube using 1 ml pipette. For this wide mouth
blue tip can be used
15.
Add 800 µl of chloroform. Mix well on end to end mixer for
10 min.
16.
Spin at 10000 rpm for 2-4 min.
17.
Transfer the upper aqueous phase to a new 1.5 ml microcentrifuge tube.
18.
Add 2 volumes of absolute ethanol or isopropanol and mix
by inverting the tube upside down for 20-30 times.
19.
A white thread like DNA may be seen as precipitate.
20.
If there is no visible precipitate seen, leave the tube at 4oC
for 30-60 min for complete precipitation
21.
Spin for 2000 rpm for 2-3 min.
22.
Discard the supernatant without disturbing the DNA pellet.
23.
Add 1 ml of 70% ethanol to wash DNA precipitate.
24.
Mix well and centrifuge at 10000 rpm for 2-4 minutes.
Discard supernatant
25.
Dry DNA precipitate by incubating at 55oC for 5 min.
26.
Dissolve in100 µl of sterile distilled water or 1X TE.
27.
Electrophores
the
DNA sample
by Agarose gel
electrophoresis.
Well 1 indicates isolated A contaminated with RNA
Well 2 indicates isolated DNA without RNA
Observation
Electrophoresis of isolated DNA sample on agarose gel results in
the separation of DNA in the distinct bands. If ethidium bromide is
used as a fluoresecent dye, isolated DNA can be visualized as orange
color band on UV trasilluminator.
Result
The isolation of genomic DNA from blood cells was done successfully.
47
Experiment No. 17
Aim: Isolation of RNA from yeast
Principle
Total yeast RNA is obtained by extracting whole cell homogenate
with phenol, the concentrated solution of phenol disturbs protein and
nucleic acid interaction thus causing denaturation of protein and
nucleic acid will be detached from the association of protein forming
aqueous layer on centrifugation. The RNA is precipitated by alcohol.
The product obtained is free of DNA but usually contaminated with
polysaccharide.
Requirements
Active dry yeast
Concentrated Phenol
Ethanol
20% Potassium acetate
Ether
Standard RNA
Agarose gel electrophoresis unit
Method
1. Mix 3 gm of activated dry yeast in 12 ml of distilled water for 15
min.
2. Add 15 ml concentrated phenol
3. Stir the solution mechanically for 30 min.
4. Centrifuge solution at 3000 rpm for 15 min.
5. Remove upper aqueous layer carefully with long tip pipette.
6. Filter the upper aqueous layer through funnel packed with glass
wool.
7. Measure the volume of filtrate and add 1 ml of 20 % potassium
acetate for every 9 ml of filtrate.
8. Add 2 volumes of ethanol (chilled) and in keep in cold condition
(-20oC) till precipitation of RNA (Approximately 2 hr).
9. Spin at 10000 rpm for 10 min to collect the pellet.
10.
Wash the pellet with alcohol: ether in 3:1 ratio for at least
three times.
11.
Wash the pellet with pure ether.
12.
Dry the pellet overnight at room temperature.
13.
Re-suspend the pellet in Tris-EDTA buffer or sterile distilled
water and electrophores by agarose gel electrophoresis.
48
Observation
Electrophoesis of isolated RNA sample by agarose gel electrophoresis
results in the separation of isolated RNA as distinct band in the low
base pair region of low base pair marker. Isolated RNA can be
estimated by Orcinol method of RNA estimation mentioned in the
laboratory manual of SBT 07.
Result
Isolation of RNA from yeast cells was performed successfully.
49
Experiment No. 18
Aim: Restriction endonuclease digestion of genomic DNA
Principle
Restriction enzyme digestions are performed by incubating doublestranded DNA molecules with an appropriate amount of restriction
enzyme, in its respective buffer as recommended by the supplier, and
at the temperature for that specific enzyme. The optimal sodium
chloride concentration in the reaction varies for different enzyme, and
a set of three standard buffers containing three concentrations of
sodium chloride are prepared and used when necessary. Typical
digestions included a unit of enzyme per microgram of starting DNA,
and one enzyme unit usually ( depending on the supplier) is defined as
the amount of enzyme needed to completely digest one microgram of
double-stranded DNA in one hour at the appropriate temperature.
These reactions usually are incubated for 1-3 hours, to ensure
complete digestion, at the optimal temperature for enzyme activity
generally at 37oC.
Generalized Procedure
1. Prepare the reaction for restriction digestion by adding the following
reagents in the listed order to a micro-centrifuge tube
Protocol
Sterile double distilled water
reaction mixture
adjustable according to the
10X Assay buffer
one-tenth volume ( 2 ml)
DNA
1 µl
Restriction enzyme
1 – 10 units per µg DNA
* If desired, more than one enzyme can be included in the digest if
both enzymes are active in the same buffer and the same incubation
temperature.
Note
The volume of the reaction depends on the amount and size of the
DNA being digested. Larger DNAs should be digested in larger total
volumes (between 50 – 500 ul ).
50
Gently mix by pipetting and incubate the reaction mixture at the
appropriate temperature (typically 7oC) for 1- hours.
Inactivate the enzyme(s) by heating at 70 – 100oC for 10 minutes or
by phenol extraction ( confirm the degree of heat inactivation for a
given enzyme)
Protocol for the restriction digestion of λ DNA
1. Prepare the reaction mixture for restriction digestion by adding
the following reagents in the listed order to a micro-centrifuge
tube.
Note: In the protocol given below volume of water to be used in the
reaction mixture has not been mentioned. It is dependant upon the
concentration of reagents supplied or used at the time of experiment.
According to the concentration of reagents used it should be adjusted.
Single digestion with Hind III
Sterile distilled water
µl
10X
3.0 µl
0.0
assay
buffer
Lambda DNA (at 0.286 µg/ µl = 5 µg DNA)
17 µl
Restriction enzyme Hind III ( 10 units/ µl =100 uints)
10 µl
Total volume
30 µl
Single digestion with E.coRI
Sterile
0.0 µl
distilled
water
10X
3.0 µl
assay
buffer
λ DNA (at 0.286 µg/ µl = 5 µg DNA)
µl
17
Restriction enzyme E.coRI ( 10 units/ µl )
10 µl
Total volume
51
30 µl
Set up of the double digestion with Hind III and E.coRI
Sterile
0.0 µl
10X
1.5 µl
10X
1.5 µl
λ
DNA
17 µl
distilled
assay
buffer
assay
(at
water
for
buffer
0.286
for
µg/
µl
Restriction enzyme E.coRI (10 units/ µl )
Restriction enzyme Hind III (10 units/ µl
E.coRI
Hind
=
5
µg
III
DNA)
5 µl
5 µl
Total volume 30 µl
The volume of water in all the reaction tubes should be adjusted
according to the reaction volume of other components of the mixture.
2. Incubate at least for 30 min at 37oC/ an hour might be Ok,
perhaps need for double digestion.
3. Halt the reaction by placing on ice, add 6 µl of 6X gel loading
dye, mix with same pipette, reset volume to 25 µl for next step.
4. Load the wells with the digested sample by placing Ladder in
first well and control DNA into second well and digested sample
in the remaining well.
52
In the above figure the numbers denoting different fragments in the
ladder indicate size of the fragment in base pairs.
Observation
On electrophoresis of the digested sample bands of fragments of λ
DNA of different sizes are observed on the agarose gel. These
fragments are compared with the control λ DNA and DNA base pair
ladder. On comparison it is observed that well in which λ DNA is
loaded shows intact band but other wells shows bands of different
sizes. These bands are compared with the DNA base pair ladder and
their sizes are determined.
Result: Restriction endonuclease digestion of the λ DNA with the
restriction endonucleases E.coRI, HindIII and mixture of E.coRI,
HindIII has been performed successfully.
53
Experiment No. 19
Aim: Analysis of DNA fragments by agarose gel electrophoresis
Principle
Electrophoresis of proteins and nucleic acids is the migration of these
biomolecules under the influence of an electric field: it is the most
important methodology of investigating genetic phenomenon at the
molecular level.
Agarose is a polysaccharide derived from seaweed, which forms
a solid gel when dissolved in aqueous solution at concentration
between 0.5 and 2.0% (W/V). Agarose used for electrophoresis is a
more purified form of the agar used to make bacterial culture plates.
Such chemically purified agarose are used chiefly for preparative
electrophoresis of DNA and for digestion of DNA with restriction
enzymes in situ. Special grades of low – gelling temperature agarose
that can be used to analyze very small fragments of DNA (10-500 bp)
are also available in the market.
Agarose gels are prepared by melting the agarose in the desired
buffer until a clear transparent solution is achieved. Melted agarose is
poured into a mold and allowed to harden. Upon hardening it forms a
matrix, when the electric field is applied across the gel, DNA that is
negatively charged at neutral pH, migrates towards the anode. Large
molecules migrate more slowly because of greater frictional drag and
because they will move slowly thorough pores of the gel less efficiently
than smaller molecules.
Super-helical circular, nicked circular and linear DNA molecules
of the same molecular weight migrate through agarose gel at different
rates i.e. under same experimental conditions Superhelical circular,
nicked circular DNA migrates faster than linear DNA.
Materials and equipments
Electrophoresis unit
Micropipettes
Microtips
Tip boxes
Autoclavable tape
Flasks
Hot plate or micro-oven or waterbath
Distilled water
Autoclave
Ethidium bromide or methylene blue
Agarose
54
Appropriate Buffer
UV transilluminator or gel documentation system
Buffers for Electrophoresis
The mobility of the DNA during electrophoresis depends on the
composition and ionic strength of the electrophoresis buffer. Two
buffers are commonly used for DNA electrophoresis: they are Trisacetate with EDTA and Tris-borate with EDTA. The pH of these buffers
is basic, therefore the DNA, because of negatively charged phosphate
backbone migrates towards anode. For smaller DNA fragments (less
than 12 to 15 kb) either the 1X TAE or TBE (1X or 0.5) is suitable for
use. For larger DNA fragments the best buffer to be used is TBE with
combination to low field strength (1 – 2 V/cm). The depth of buffer
over the gel in horizontal electrophoretic system should be 3 –5 mm.
Less buffer may cause dry out during electrophoresis whereas excess
buffer depth may cause decreased DNA mobility and eventually
distortion of bands.
Buffer depletion is also an important parameter for selection of
the buffer system. Depletion rate is influenced by the type and
buffering capacity of buffer used. Buffer depletion is checked by gel
melting, smearing of DNA and or overheating of chamber. In such
instances buffer is recirculated intermittently. Tris-phosphate buffer is
also used for DNA electrophoresis. Like TBE buffer, TPE buffer has a
high buffering capacity but DNA recovery is difficult. This buffer is
suitable when recovered DNA is used in a phosphate sensitive
reaction.
Table: Composition of buffers for electrophoresis
Buffer
Working solution
Concentrated stock solution (
per litre)
Tris-acetate 1X:
0.04
M
Tris- 50 X: 2.42 g Tris base
(TAE)
acetate
57.1 ml glacial acetic acid
0.001 M EDTA
100 ml 0.5 M EDTA (pH
8.0)
1X:
0.09
M
Tris- 10X: 108 g Tris base
Trisphosphate
15.5
ml
85%
w/v
phosphate
( TPE)
0.002 M EDTA
phosphoric acid
40 ml 0.5M EDTA ( pH
8.0)
0.5X: 0.045 M Tris- 5X: 54 g Tris base
Tris-borate
27.5 g boric acid
(TBE)
borate
20 ml 0.5 M EDTA (pH 8.0)
0.001 M EDTA
Alkaline
1X: 50 mN NaOH
1X: 5 ml 10 N NaOH
1 mM EDTA
2 ml 0.5 M EDTA ( pH 8.0)
55
Concentration of agarose used for electrophoresis
The optimal concentration of agarose depends on the size of the DNA
fragments to be resolved. Agarose grade is also important parameter
in such separation. Following table gives the idea about percentage of
agarose used for the separation of molecules of DNA of variable sizes.
Table: Agarose concentration and range of separation of DNA
molecules
Agarose % in
Buffer
(W/V)
0.3
0.6
0.7
0.9
1.2
1.5
2.0
Range of
separation of
linear DNA
fragments ( kb)
5 – 60
1-70
0.5 – 10
0.5 – 7.0
0.4 – 6.0
0.2 – 3.0
0.1 –2.0
Optimal Voltage and Electrophoretic times
The distance between the electrodes is used for the determination of
the voltage gradient, not the gel. Voltage is too high, band streaking
may result whereas too low voltage may result in broadening of the
bands due to dispersion and diffusion.
Optimal voltage is required to achieve sharp bands. The gel
should be run until the band of interest migrates 40 – 60 % down the
length of the gel. For various agarose gel systems the optimal voltage
to run DNA is recommended as given in the following table.
Size
Voltage
Recovery
< or = 1 kb
1to 2 kb
> 12 kb
5 Vcm-1
4 – 10 Vcm-1
1 –2 Vcm-1
TAE
TAE
TAE
Buffer
analytical
TBE
TAE, TBE
TAE
Gel loading buffers
Gel loading buffer serve three purposes in DNA electrophoresis
• Increase the density of the sample and ensure even application of
DNA into every well.
• Add color to the sample and thereby simplify loading
• Add mobility dyes, which enable one to monitor the rate of
electrophoresis process.
56
Loading buffers are prepared in 6 to 10 ford concentrated solutions.
For performing alkaline gel electrophoresis alkaline phosphate buffer
is used. To increase the sharpness of band ficoll polymer is used as a
sinking agent instead of glycerol.
Composition and storage temperature of loading buffer
Loading buffer
6X solution (w/w)
Storage
temperature
Sucrose based
40% Sucrose
4oC
0.25% Bromophenol Blue
0.25% Xylene Cyanol FF
Glycerol based
30% Glycerol in distilled 4oC
water
0.25% Bromophenol Blue
0.25% Xylene Cyanol FF
®
Ficoll based
15% Ficoll (type 400) Room
temperature
polymer in
distilled water
0.25% Bromophenol Blue
0.25% Xylene Cyanol FF
4oC
Alkaline
6 mM EDTA
0.15%
Bromophenol
Green
0.25 Xylene Cyanol FF
Preparation of Agarose gel
1. Seal the open ends of the plastic tray supplied with
electrophoresis apparatus with autoclavable tape. The tray
should be clean and dry. Keep the tray on horizontal bench.
2. Prepare sufficient TAE buffer to fill the electrophoresis tank and
to make gel.
3. Add correct quantity of pure powdered agarose to a measured
quantity of buffer in a conical flask. The buffer should not exceed
in volume more than 50% of the size of the flask. Heat the
mixture in a boiling water bath or in a microwave oven or on hot
plate
until
agarose
completely
dissolves
forming
clean/transparent solution.
4. Cool the solution to about 55 – 60oC. If planned add Ethidium
bromide to a final concentration of 0.5 µg/ml and mix
thoroughly.
5. Position the comb 0.5 – 1.0 mm above the plate so that a well is
formed when agarose is added.
57
6. Pour the warm agarose in the mold / gel plate. The gel should be
3 –5 mm thick. Avoid air bubbles in the setting gel.
7. After the gel is completely set (30 – 45 min at room
temperature), carefully remove the comb.
8. Add just enough electrophoresis buffer 2- 3 mm above the gel.
9. Mix the sample of DNA with the desired gel-loading buffer.
Slowly load the mixture onto the slots / wells of the submerged
gel using a disposable micro tips with micropipettes.
10.
Close the lid of the set tank and attach the electric leads to
the power pack. The connection to the tank should be given in
such a way that DNA sample will migrate towards anode (red
lead). Apply required voltage. As the electrophoresis starts we
can see the bubbles generated at the anode and cathode within
few minutes. The bromophenol blue should migrate from wells
into the body of the gel. Allow the separation / run until the
bromophenol blue have migrated the appropriate distance on
gel.
11.
Put off the electric supply and remove the leads and lid
from the gel tank. In case the ethidium bromide was present in
the gel and electrophoresis buffer, examine gel by UV light and
photograph otherwise, stain the gel with ethidium bromide for
20 – 30 min in a tank and then observe the bands. Alternatively
DNA can be detected by keeping the gel in the solution of
methylene blue dye (0.02%) in distilled water for 15 min.
Destain the gel for 15 min in distilled water.
Photograph of Agarose Gel Electrophoresis apparatus
58
Observation
Observe the agarose gel after electrophoresis and compare the bands
obtained on the gel with DNA base pair ladder and/or control DNA and
determine the size of the band obtained.
Result
The given sample of the DNA has been analyzed by agarose gel
electrophoresis and the band(s) obtained was found to be of ------------------ size(s)
59
Experiment No. 20
Aim: Isolation of Plasmid DNA from bacteria by alkaline lysis
miniprep method
Principle
Plasmids are double stranded circular, self replicating extra
chromosomal DNA molecules. They are commonly used as cloning
vector in molecular biology. Many methods are used to isolate plasmid
DNA, essentially involving the following steps.
•
•
•
Growth
Harvest and lysis of bacteria
Isolation of plasmid DNA
Alkaline lysis method for rapid purification of plasmids exploits the
topological difference between plasmid circles and linear chromosomal
fragments. Cells are lysed by treating with alkali (NaOH) and a
detergent, sodium dodecyl sulphate (SDS). SDS denatures bacterial
proteins and NaOH denatures the plasmid and chromosomal DNA.
However, in the case of plasmids the strands remain closely
circularized since they are linked by the intertwined backbones of
double helix. In contrast, strands of linear/nicked DNA are free to
separate completely. When this mixture of plasmid and chromosomal
DNA is neutralized by the addition of sodium acetate, renaturation
occurs. Renaturation of plasmid is rapid and accurate since the strands
are already in close proximity. Linear molecule generated by random
shearing of chromosomal DNA renature less accurately forming
networks of DNA that can be removed from lysate by centrifugation,
together with denatured protein and RNA. Plasmid DNA remains in
solution and can be precipitated using ethanol/isopropanol.
Agarose gel electrophoresis separates plasmid DNA by its size
and shape. A preparation of plasmid DNA has predominantly super
coiled form that runs faster by virtue of its compact structure.
Introduction of single/double strand breaks leads to presence of
relaxed and linear forms respectively. These forms run more slowly
since their open structure experience more resistance passing through
the gel matrix. These are seen as bands above the super coiled form.
However the presence of linear form is very rarely seen in a plasmid
preparation.
60
Reagents
Buffer P1:
Tris Base (50 mM pH 8)
: 6.055 gm
EDTA.2H2O (10 mM )
: 3.722 gm
100 µg /ml RNase A ( Add after autoclaving) : 100 mg
Dissolve the Tris base and EDTA in 800 ml of distilled water. Adjust the
pH to 8 with HCl. Autoclave this solution. Allow the solution to cool to
room temperature before making final adjustment to pH. Add RNase A
at the time of isolation and adjust the volume to 1 L with sterile
distilled water.
Note: Above composition is for 1 L but it is suggested that solutions
should be prepared as per the need of the practical.
Buffer P2 (Lysis buffer):
NaOH (200mM)
: 8.0 gm
Sodium dodecyl sulphate: 10 gm
Dissolve above chemicals in 900 ml autoclaved distilled water and
adjust the volume to 1 L with sterile distilled water. Store this reagent
at room temperature.
Buffer P3 (pH 5.5):
Potassium acetate (3M): 294.45 g
Dissolve above chemical in 500 ml of sterile distilled water and adjust
the pH to 5.5 with glacial acetic acid. Adjust the volume to 1 L with
sterile distilled water.
Ethanol or isopropanol
Agarose
Electrophoresis assembly
Method
1. Inoculate the bacterial culture from which plasmid DNA is to be
isolated in Luriea-Bertani broth. If plasmid codes for antibiotic
resistance then that antibiotic must be added to the LB broth to
amplify the plasmid DNA. If it is DH5α strain of E. coli containing
pUC18 plasmid then Ampicillin at a concentration of 100 ug /ml
is added.
61
2. Incubate culture overnight at optimum temperature of bacteria
(e.g 37oC in case of DH5α strain of E. coli containing pUC18
plasmid.).
3. Pipette 2 ml culture in two 2 ml microfuge tube.
4. Spin at 6000 rpm for 8-10 min. discard the supernatant and
invert the vial in blotting paper to drain the left over
supernatant. Place on ice crystals.
5. Add 300ul of ice cold Buffer P1 and 300 ul of Buffer P2 in cell
pellet.
6. Mix by inversion
7. Incubate both the tubes for 5 min at room temperature
8. Add 300 µl of Buffer P3.
9. Mix by inversion.
10.
Incubate on ice for 15 min.
11.
Centrifuge at 16000 rpm for 30 min
12.
Take supernatant and measure it.
13.
Add 0.7 volume of isopropanol or ice cold ethanol to the
measured supernatant.
14.
Incubate at room temperature for 2 hr if isopropanol is
used and if chilled ethanol is used incubate at intense cold
condition for 2 hr or overnight.
15.
Centrifuge at 16000 rpm at 25oC for 30 min.
16.
Decant the supernatant. Invert the vial on blotting paper
to drain out left over supernatant. DNA will be seen as a white
precipitate, sticking to the sides of the wall.
17.
Dry the sample at 37oC for 10 to 15 min till there is no
trace of ethanol or isopropanol
18.
Resuspend the dried plasmid DNA in sterile distilled water
or TE buffer.
19.
Check isolated resuspended sample on agarose gel
electrophoresis for successful isolation of plasmid DNA.
62
Observation
Isolated plasmid DNA is electrophoresed on 1% agarose gel. Extracted
sample is compared with control DNA (if control is available for e.g. in
case of pUC18 plasmid). Usually two bands corresponding to super
coiled and nicked forms are seen on gel. Observation is also done for
the presence or absence of RNA in sample as RNase is added in the
Buffer P1 to remove RNA contamination. Out of two bands seen on the
agarose the band which moves faster is supercoiled plasmid DNA and
another band above the supercoiled plasmid DNA band is the nicked
plasmid DNA. The band which moves faster than and is observed far
distant from the supercoiled plasmid DNA is RNA molecule because
RNA is smaller molecule and migrate faster on agarose gel.
Result
Plasmid DNA has been isolated successfully from bacteria by alkaline
lysis miniprep method
63
Experiment No. 21
Aim: Preparation of competent cells
Principle
Bacterial transformation is the process in which bacteria manage to
uptake or bring in free/ external DNA from the environment. The
purpose of this technique is to introduce a foreign plasmid DAN into
the bacteria and to use these bacterial to amplify the plasmid DNA.
The ability to take up DNA efficiently by most bacteria is limited
in nature. However bacterial cells can be artificially induced to take up
DNA by treating them with CaCl2. Culture of such cells that are capable
of taking up DNA is said to be competent. The conditions required to
produce competence vary from species to species.
The phenomenon of competence has not been understood very
well. It appears to result from changes in cell wall of bacteria and is
probably associated with the synthesis of cell wall. In the course of
developing competency, receptors of same kind are either formed or
activated on the cell wall, which are responsible for initial binding of
DNA. The complex thus formed are resistant to DNases .
Requirements
Luria Bertani Broth
E. coli DH5α strain
Micropipettes
Petriplates
Micro centrifuge tubes
CaCl2
Ice crystals
Incubator cum shaker
Handling of the reagent
1. All the tubes and reagents used for competent cell preparation
should be pre-chilled
2. The reagents and tubes provided are sterilized and should be
handled with care under aseptic condition.
3. Prepare 0.1 M CaCl2 freshly from 1 M stock as and when
required.
Method
1. Revival of the culture: Streak a loopful of E. coli DH5α strain
from the stock on a fresh Luria- Bertani ( LB) medium plates to
get isolated colonies. Incubate the plate overnight (16-20 hr) at
37oC.
64
2. Pick a single colony from the plate and transfer it into 5 ml LB
broth. Incubate the culture overnight (16-20 hr) at 37oC.
3. Transfer the 5 ml overnight grown culture in 500 ml L B broth.
Incubate the culture at 37oC with continuous shaking at 100150 rpm till the OD600 reaches 0.23 to 0.26 (Determine the OD600
every 20 – 30 min). Cary out entire procedure in sterile
condition.
4. Once the required OD is attained quickly chill the culture on ice
and leave it on ice for 10 – 20 min.
5. Transfer the culture aseptically into sterile centrifuge tubes and
spin at 4000 to 6000 rpm for 10 min.
6. Decant the supernatant and resuspend the pellet in 6 ml of ice
cold 0.1 M CaCl2. Keep on ice for 30 min.
7. Centrifuge at 4000 to 6000 rpm for 10 min at 4oC.
8. Decant the supernatant and resuspend the pellet in 0.5 ml of ice
cold 0.1 M CaCl2.
9. In this way after giving the treatment as above the bacterial
cells develop competence and cells are said to be competent.
10.
Using a sterile pipette tip, transfer 100 ul of above
suspension of the competent cells to a chilled, sterile micro
centrifuge tubes kept on ice
11.
Carry out the transformation of the competent cells
prepared in this way with pUC18 plasmid DNA.
Note: Transformation procedure has been described in the next
experiment.
Observation
The E. coli DH5α strain of bacteria after subjecting to the above
treatment develops competence and become competent for the uptake
of foreign DNA e.g pUC18 plasmid DNA. Whether the competence has
been developed or not can be checked by comparing the competent E.
coli DH5α strain with control E. coli DH5α strain which has not
undergone above cold CaCl2 treatment. If the cells of E. coli DH5α
strain have become competent it will uptake the pUC18 plasmid DNA
and will show expression of desired trait, which is coded for by pUC18
plasmid for e.g. antibiotic resistance or β- galactosidase production.
Competent cells plated on the antibiotic containing plate will survive
whereas cells, which have not been transformed, will not grow on
antibiotic containing plate.
65
Result
Preparation of competent cells E. coli DH5α strain by cold CaCl2
treatment has been done successfully.
Note: Result written above is a prototype and should be copied by the
student only after getting the desired result. If desired result is not
obtained negative result should be reported and experiment should be
performed again.
66
Experiment No. 22
Aim: Transformation of bacterial cell with foreign DNA
Principle
Bacterial transformation is the process in which bacteria manage to
uptake or bring in free/ external DNA from the environment. The
purpose of this technique is to introduce a foreign plasmid DAN into
the bacteria and to use these bacterial to amplify the plasmid DNA.
The competent cells are exposed briefly to a temperature of 42oC
– “heat shock”, wherein pores are created and DNA is taken up.
Immediately chilling on ice ensures closer of pores, cells are then said
to be transformed. These cells are then screened for transformants
and recombinants.
If pUC18 plasmid is used as foreign molecule for transformation
then following discussion is applicable.
Screening of transformants:
Selection of cells containing foreign DNA is done based on the selection
of marker carried by this DNA e.g. pUC plasmid has ampicillin resistant
factor that enables only transformed cells to grow on LB-ampicillin
plates. Non-transformants, which are ampicillin sensitive, do not
produce colonies on the selective medium. Transformants and nontransformants are therefore easily distinguished.
Screening of recombinants
Identification of recombinants among the transformed cells is generally
done by insertional inactivation. With most cloning vector, insertion of
DNA fragments into the plasmid destroy the integrity of one of the
genes present on the molecule. As a result the characteristic coded by
the inactivated gene is no longer displayed by host cells and this is
called insertional inactivation. pUC is a high copy number plasmid
of size 2686 bp with col E1 origin of replication. It carries 54 bp
polycloning region and ampicillin resistant marker, along with coding
information for the first 146 amino acid (amino terminal) of β galactosidase (LacZ) gene. Some strains of E.coli. bear a deletion at
the amino terminal end of LacZ gene and thus synthesize an inactive
terminal fragment. On transforming such competent bacterial strain
with pUC18 the host and plasmid encoded fragment associate to form
an enzymatically active protein. This type of complementation is
known as α-complementation. Lac+ bacteria that result from αcomplementation can be recognized as they form blue colored colonies
in the presence of X-gal ( chromogenic substrate for β-galactosidase
and IPTG ( inducer for the expression of the enzyme). However
67
insertion of fragment of foreign DNA into the polycloning site of
plasmid results in production of an amino-terminal fragment that is not
capable of α-complementation. Hence cells carrying recombinant
plasmid will form white colonies. This is also referred to as blue white
screening.
Requirements
Materials to be stored at –20oC
Bacterial Host for e.g. E. coli DH5α strain
IPTG (isopropyl -β-D-thiogalactopyranoside)
X-gal ( 5-Bromo-4-Chloro-3-Indolyl-β-D-galactoside)
Materials to be stored at 4oC
Plamid DNA ( pUC18)
Calcium chloride 1M stock
Ampicillin
Materials to be stored at room temperature
Agar
LB broth
Micro centrifuge tubes 1.5 ml
Micropipettes
Microtips
Tip box
Shaker cum incubator
Rocker
Gloves
Pre-preparation for experiment
1. Prepare at least 5 tubes each containing 5 ml LB broth.
Autoclave these tubes of LB broth.
Prepare 2% LB broth in 25 ml distilled water and distribute 5 ml
in each of the 5 tubes and autoclave.
2. Prepare two 500 ml LB broth flask and autoclave it.
3. Autoclave 1.5 ml micro centrifuge tubes, 10 µl, 200 µl and 1000
µl micro tips with their tip boxes, distilled water, petriplates.
4. X-Gal (20 mg/ml) Dissolve 20 mg X-gal ( 5-Bromo-4-Chloro-3Indolyl-β-D-galactoside) in 20 ml N,N’ – dimethyl formamide and
store at -20oC in light protected tube.
5. 0.1 M IPTG – 5 ml: Dissolve 0.119g IPTG (isopropyl -β-Dthiogalactopyranoside) in 4 ml sterile distilled water and adjust
the volume to 5 ml with sterile distilled water. Store at -20oC.
6. Preparation of LB + Ampicillin + IPTG + X - gal plate
68
Add 2 g agar and 2 g LB broth to 100 ml of distilled water.
Autoclave, cool to 50oC and add Ampicillin at a concentration of
100 µg/ml, 20 µl of IPTG and 500 µl of X-gal
Make Ampicillin solution fresh by dissolving required quantity in
sterile water.
Prepare LB + Ampicillin + IPTG + X - gal plates on the date of
inoculation.
Method
Note: This experiment is in continuation with the previous experiment
hence preparation of competent cells has not been written here.
1. Prepare competent cells as described in the previous
experiment.
2. Label one tube containing 100 µl of competent cells as ‘+’ (with
plasmid) and another as ‘-’ ( without plasmid).
3. Add 5 µl of pUC18 plasmid to the tube labeled ‘+’. Tap the tube
with your finger to mix the contents.
4. Place both the tubes on ice for 20 minutes.
5. When the incubation period on ice is nearly over prepare a water
bath at 42oC.
6. Heat shock the bacteria by keeping the vial in 42oC waterbath
for exactly 90 seconds.
7. Immediately return the tubes after heat shock to the ice and let
them stand on ice for additional 10 min.
8. Add 1 ml of LB medium to each tube and transfer the contents to
a culture tube.
9. Incubate the culture for 45 min at 37oC on rocker. This step is
essential to allow the bacteria to recover from the heat shock
treatment and to express the antibiotic resistance marker
encoded by the plasmid.
10.
From the tube labeled ‘+’ from step 7, transfer 50 µl of
culture to separate LB + Ampicillin + IPTG + X - gal plate. Also
transfer 50 µl of culture from the tube labeled ‘ – ’ from step 7
as a control to a separate LB + Ampicillin + IPTG + X - gal
plate. Spread thoroughly using a spreader.
11.
Incubate the plate overnight at 37oC.
12.
Observe the plate after 24 - 48 h.
69
Photograph showing transformed colonies on LB+ Ampicillin s + X-Gal
+ IPTG plates
Observation
LB + Ampicillin + IPTG + X – gal plate inoculated with ‘+’ culture
will show blue colored colonies whereas plates inoculated with ‘-’
culture will not show any colony. This indicates the successful
transformation of E. coli DH5α strain with pUC18 plasmid DNA.
Result
The DH5α strain of E. coli has been transformed with pUC18
plasmid DNA successfully.
70
Experiment No. 22
Aim: Filing of Patent with example
Definition
A patent is the right granted by a government to an inventor to
exclude others from imitating, manufacturing, using or selling the
invention in question for commercial use for specified period.
OR
Patent is an official right granted to a person or institution to make,
use or sell a product or an invention.
Biopatent
“Biopatent is a government protection to an inventor of a biological
material, giving him the rights of manufacturing, using and selling the
invention for a specific period.”
Biopatents are awarded for
1) Inventing new biological products, their production techniques
and applications.
2) For the discovery of new strains of microorganisms.
3) For the genetically modified transgenic plants and animals.
4) For the synthesis and discovery of novel DNA sequences.
5) For proteins formed by various DNA sequences.
6) For discovery of cell lines and new biotechnological methods.
Addresses Of Indian Patent Design & Trademarks Offices
For Patent & Design related Work Contact the following Addresses:
O/o Controller General of Patents Designs & Trade Marks,
CGO Building,
101 M K Road
Mumbai 400 020
India
Phone No.: 91-22-201 7368
: 91-22-203 9050
Fax No.: 91-22-205 3372
71
Regional Patent Offices Address:
Eastern Region: CALCUTTA
Dr. S K Pal,
Asst. Controller of Patents & Designs,
Nizam Palace,
2nd MSO Building (5th to 7th Floors)
234/4 Acharya Jagadish Bose Road,
Kolkata 700 020 India
Phone: 91-33-247 4401-03
91-33-247 3851
91-22-240 1353
Fax : 91-33-247 3851
Northern Region : DELHI
Shri K.S. Kardam,
Asstt. Controller of Patents & Designs,
Patent Office Branch,
W-5, West Patel Nagar,
New Delhi 110 008
Phone: 91-11-5871255/6/7/8
91-11-5871245
91-11-5876209
Fax : 91-11-5872532
Email: delhipatent@vsnl.com
Western Region : MUMBAI (BOMBAY)
Shri N K Garg,
Assistant Controller of Patents & Designs,
Todi Estates, 3rd Floor,
Sun Mill Compound,
Lower Parel(West)
Mumbai 400 013 India
Phone: 91-22-492 5092
91-22-492 4058
Fax : 91-22-495 0622
Southern Region : CHENNAI (MADRAS)
Shri M S Venkatraman,
Assistant Controller of Patents & Designs
72
Wing 'C' (C-4, A), III Floor, Rajaji Bhavan
Besant Nagar,
Chennai 600 090 India
Phone: 91-44-490 1495
91-44-490 1496
Fax : 91-44-490 1492
SOUTHERN REGION CHENNAI (MADRAS)
Shri M H Mahendra,
Deputy Registrar of Trade Marks,
Trade Marks Registry,
Rajaji Bhavan,
2nd Floor, "D" Wing
Besant Nagar,
Chennai, India
Phone: 91-44-490 2791
91-44-490 2787
Fax : 91-44-490 2787
FOR PATENT FULL TEXT & SEARCH CONTACT
The Senior Documentation Officer,
Office of the Patent Information System,
3rd Floor
Block 'C' CGO Complex,
Seminary Hills, Nagpur 440 006 MS, India
FOR OVERALL FUNCTIONS
Dr. P P Paranjpe
Patent Information System
3rd Floor,
Block 'C', CGO Complex
Seminary Hills
Nagpur 440 006
India
Phone : 91-712-511241
91-712-510670
Fax : 91-712-510186
E-mail: pisnagp@nag.mah.nic.in
The above Information is extracted from Minstry of Industry
web site
73
Technology Information Forecasting Assessment
Council(TIFAC)
An
autonomous organization under Department of Science &
Technology,
Government of India, is working in the field of Technology
information,
Forecasting for the last one decade. It has a Patent Facilitating
Cell, which is
very active in patent related activities in India, also publishing
a newsletter
(Intellectual Property Rights Bulletin), advisory Services, Case
studies. The
Contact address is: Patent Facilitating Cell
Technology Information, Forecasting & Assessment Council
TIFAC
Technology Bhawan
New Mehrauli Road
New Delhi - 110 016
India
Phone : 91-11-6859581
91-11-6863877
91-11-6967458
Fax
:
91-11-6863866
E-mail: tifac@nda.vsnl.net.in
Forms for filling of patent
Note: For filing of patent various forms are filled with the help of
authorized patent agent. Here only one form has been given for
practicing. Students are expected to fill this form and attach to the lab
record. It is expected from the students that they should fill the other
relevant forms supplied in this manual related to filing of patent. It is
also suggested that students should visit the official web site of
government of India www.indianpatent.nic.gov.in.
74
FORM 1
THE PATENTS ACT, 1970
(39 of 1970)
APPLICATION FOR GRANT OF A PATENT
(See sections 5(2), 7, 54 and 135; rule 39)
1. Repeat the columns
(a) to (c) if there are
more
than
one
applicants.
1. I/We, 1..
(a) 2
_____________________________
_______
(b) 3
_____________________________
_______
(c) 4
_____________________________
_______
2. Insert the name in
full. The family or
principal name in the
beginning
if
the
applicant is a natural
person.
(a) 2
_____________________________
_______
(b) 3
_____________________________
_______
(c) 4
_____________________________
_______
(a) 2
_____________________________
_______
(b) 3
_____________________________
_______
(c) 4
_____________________________
75
_______
3. Insert the complete
addresses
including
postal
index
number/code
and
state and/or country.
2. hereby declare -
(a) that I am/We are in possession of an
invention
titled
_____________________________
____
___________________________
___________
___________________________
___________
4. Insert the nationality.
(b) that
the
provisional/complete
specification relating to this invention
is filed with this application.
(c) that there is no lawful ground of
objection to the grant of a patent
to me/us
5. Repeat the columns
(a) to (c) if there are
more
than
one
inventor.
3. further declare that the inventor(s) for
5
the
said
invention
is/are
__________________________
6. Insert the name in
full.
Family or principal
name
in
the
beginning.
(a)
6.
________________________________
____
_________________________________
___
(b)
7.
________________________________
____
7. Insert the Complete
address including the
postal
code,
state
_________________________________
___
(c)
8.
________________________________
____
76
postal
code,
state
and/or country.
________________________________
____
8. Insert the nationality.
4.
9. Repeat the columns
(a) to (c) if there are
more
than
one
applications.
(a).
10.
________________________________
_____
10.
Name
country.
of
the
I/We, claim the priority from the
application(s)
filed
in
convention
countries, particulars of which are as
follows:
9.
__________________________
(b).
11.
________________________________
_____
(c).
12.
________________________________
_____
11. Application number
(d).
13.
________________________________
_____
12. Date of application.
(e).
14.
________________________________
_____
and declare that above application or each
of the above applications was the first
application(s)
in
a
convention
country/countries in respect of my/our
invention..
5. I/We state that the said invention is an
improvement in or modification of the
invention, the particulars of which are
as follows and of which I/We are the
applicant/patentee:
13.
Applicant
in
convention country.
14.
Title
of
the
invention
in
the
convention country
15. Application number
or patent number.
(a).
15.
________________________________
_____
(b).
16.
________________________________
77
_____
16. Date of application
or date of patent.
17. Application number
including
published
serial number, if any.
6.
I/We state that the application is
divided out of my/our application, the
particulars of which are given below and
pray that this application deemed to
have
been
filed
on
_______________________
under section 16 of the Act.
18. Date of filing of
provisional
specification
and/or
complete
specification.
(a).
17.
________________________________
_____
19. Complete address
including postal index
number/code
and
state
along
with
Telephone
and
Telefascimile
number(s)
20. Repeat the columns
(a) to (c) if necessary
7. That I am/We are the assignee or legal
representative of the true and first
inventors.
(b).
18.
________________
_________________
and
8. That my/our address for service in India
is
as
follows:
19.
______________________________
________________________________
________
21. Signature of the
true
and
first
inventor(s)
or
applicant
in
the
convention
country
with date.
Name of the natural
person should also be
given
below
the
signature.
________________________________
________
9. Following declaration was given by the
inventor(s) or applicant(s) in the
convention country:
I/We the true and first inventors for this
invention or the applicant(s) in the
convention country declare that the
applicant(s)
herein
is/are
my/our
assignee or legal representative.
20.. .
(a)
6..
13.
________________________________
78
(b)
7..
________________________________
_
(c)
8.
_________________________________
( -----------------------------------21
10. That to the best of my/our knowledge,
information and belief the fact and
matters stated herein are correct and
that there is no lawful ground of
objection to the grant of patent to
me/us on this application.
11. Following are the attachments with the
application:
(a) Provisional/complete specification (3
copies)
(b) Drawings (3 copies)
(c) Priority documents(s)
(d) Statement and Undertaking on
Form-3
(e) Power of Authority
(f) ………………………………………..
(g) ………………………………………..
(h) ………………………………………..
(i) Fee
Rs.
……………
in
Cash/Cheque/Bank Draft bearing No.
………… date …………….
on …………………………… Bank.
I/We request that a patent may be
granted to me/us for the said invention
22. To be signed by the
applicant(s) or by his
authorised
patent
agent.
23. Name of the natural
person
who
has
signed.
Dated this………… day of ……………. 20
Signature..
22.
..
(-------------------------------------- ) ..23..
To
The Controller of Patents,
79
)
The Patent Office,
At ………………………………………………….
……………………………………………………..
Note:
(a) Strike out which ever is inapplicable
(b) For fee: see First Schedule
80
Experiment No. 23
Aim: Patent search on internet
Extraction of information about the patents granted by various
authorized agencies to the individual, company, institution or
organization from the Internet is called patent search on the internet.
There are various governmental and nongovernmental web sites on
which patent search can be accomplished.
For e. g. www.indiabigpatents.org
www.internationalpatent.org
www.patentoffice.nic.in
On this patent search is very easy
Open this web site on the internet
Chose the option for searching of patent. Patent can be searched by
name by putting the name of the individual or company or institution
to which patent has been granted, in the window provided and clicking
search key or patent can be searched by putting the patent number in
the search window.
Observation
On searching on the internet it has been found that the patent for the
------------------------------------------------- has been granted/ not
granted/ or it is under processing. If granted then its title is ---------------------------------------------------------------------------------------The name of inventor is ---------------------------------------------------------------------------the number is ---------------------------------------- the date of application is ----------------the date of granting is -------------------------------------Result
Patent search on the internet for the -----------------------------------------------------------------------------has
been
performed
successfully.
81