Immunology RDNA Bioinformatics Manual

Lab in Immunology,
rDNA Technology and

Bioinformatics Manual
Dr.B.Prakash.,Ph.D
Head of the Department
Department of Biotechnology
PGP College of Arts &
Science, Namakkal
Lab in Immunology, rDNA Technology and Bioinformatics Manual
Ex.No:
Date :
LABORATORY SAFETY
Safety in the Laboratory Should always is in your mind. Throughout this manual
Safety recommendations are given, below are some general consideration that anyone in a
laboratory should know.
GENERAL LABORATORY SAFETY PRECAUTION
1.
Follow all instructions carefully. Use special care when you see the word
CAUTION in your laboratory instructions. Follow the safety instructions given by
your teacher.
2.
Determine the location of Fire Extinguishers, Chemical safety showers and Eye
washers, Chemical Spill Kits, and alternative exit routes for lab evacuation.
3.
Remember that smoking, eating, or drinking in the lab room is totally prohibited.
4.
Wear lab aprons when working with chemicals, hot material, or preserved
specimens.
5.
Wear safety goggles when using dangerous chemicals, hot liquids, or burners.
6.
Any chemicals spilled on the hands or other parts of the skin should be washed off
immediately with a plenty of running water.
7.
If you have an open skin wound, be sure that it is covered with a water proof
bandage.
8.
Never work alone in the laboratory.
9.
Keep your work area clean & dry.
10.
Turn of all electrical equipment, water, and gas when it is not in use, especially at
the end of the laboratory period.
11.
Tie back long hair.
12.
Report all chemicals spills or fluids to your instructor immediately for proper clean
up.
Special precautions for working with heat or fire:

1.
Never leave a lighted Bunsen burner of hot object unattended. When an object is
removed from the heat & left to cool, it should be placed where it is shielded from
contact.
Head of the Department Department of Biotechnology PGP College of Arts & Science,
Namakkal
2.
Inflammable liquid bottles should not be left open, not dispensed near a naked
flame, hot electric element or electric motor.
3.
Use test tube holders to handle hot laboratory equipments.
4.
When you are heating something in a container such as a test tube, always point
the open end of the container away from yourself & others.
5.
Use only Pyrex glasswares for heating.
6.
Allow hot materials to cool before moving them from your lab station.
7.
Make sure that Bunsen burner hoses fit tightly.
Special precautions for working with chemicals

1.
Never taste or touch substances in the laboratory without specific instructions.
2.
Never smell substances in the laboratory without specific instructions.
3.
Use materials only from containers that are properly labeled.
4.
Wash your hand after working with chemicals.
5.
Do not add water to acid. Instead, dilute the acid by adding it to water.
6.
Mix heat generating chemicals slowly.
Special precautions for working with electrical equipment

1.
Make sure the area under & around the electrical equipment is dry.
2.
Never touch electrical equipment with wet hands.
3.
Make sure the area surrounding the electrical equipment is free of flammable
materials.
4.
Turn off all power switches before plugging an appliance into an outlet.
Special Precaution for working with Glasswares and other laboratory equipments
1.
Become familiar with the names and appearance of all the laboratory equipments
you will use.
2.
Never use broken or chipped glassware.
3.
Make sure that all glasswares are clean before you using it.
4.
Do not pick up broken glass with your bare hands. Use a pan and a brush.
5.
If a Mercury thermometer breaks, do not touch the mercury. Notify your teacher
immediately.
6.
Do not aim the mirror of your microscope directly at the sun. Direct sun light can
damage the eyes.
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Use care handling all sharp equipments, such as scalpels and dissecting needles.
7.
Special precautions for working with live or preserved specimens

1.
If live animals are used treat them gently. Follow instructions for their proper care.
2.
Always wash your hands after working with live or preserved organisms.
3.
Specimens for dissection should be properly mounted and supported. Do not try to cut
a specimen while holding it in the air.

4.
Do not open Petri dishes containing live cultures unless you are directed to do so.
5.
Detergents (Dettol 5 10%) should be used to sterilize and clean benches, glassware
and equipment.
6.
Safety cabinet should be used while working with microbes.
7.
Lab coats should be worn during the work in the lab.
8.
Disposable items should be collected and autoclaved.
First Aid
8.
Injuries: bleeding should be reduced using bandages; the wound should be cleaned
with iodine alcohol mixture, and wrapped with sterile bandage.

9.
Acid and fire burns: body burns must be washed immediately with tap water. Eye
burns must be washed using eye washer, special cream for burns can be used.
10.
Poisoning: if any toxic chemical is swallowed, the mouth must be sensed with water,
in case of acid, milk is drunk, in case of alkaline, diluted acetic acid ( vinegar) can be used.
11.
Skin contamination requires washing with water and removal of contaminated
clothing, if the contaminant is insoluble in water remove with soap and water.
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Ex.No:
Date :
SEPERATION OF PLASMA AND SERUM FROM BLOOD

AIM:
To separate the serum and plasma from the given blood samples
INTRODUCTION:
Blood plasma is the liquid component of blood in which the blood cells are suspended. It
makes about 60% of the total blood volume. It is composed of mainly water (90% by
volume), and contains dissolved proteins, glucose, clotting factors, mineral ions, hormones
and carbon di oxide (plasma being the main component for excretory product transportation).
Plasma is the supernatant fluid obtained when the anti-coagulated blood has been centrifuged.
The blood is mixed with an appropriate amount of anti-coagulant like heparin, oxalate or
EDTA. This preparation should be mixed immediately and thoroughly to avoid clotting.
Blood serum is blood plasma without fibrinogen or other clotting factors.
PROCEDURE:
Blood Plasma Preparation
1. Draw blood into centrifuge tube containing approximately 1.8mg of potassium EDTA
per ml blood. Be sure to take the full volume to ensure the correct blood to anticoagulant ratio.
2. Invert centrifuge tubes 10 times carefully to mix the blood and anti-coagulant and
store at room temperature until centrifugation.
3. Samples should undergo centrifugation immediately. This should be carried out for a
minimum of 10min at 1000-2000rcf (generally 1300rcf) at room temperature. Do not
use breaks to stop the centrifuge.
4. This will give three layers from top to bottom plasma, leucocytes (Buffy coat), and
erythrocytes.
5. Carefully aspirate the supernatant (plasma) at room temperature and pool in a
centrifuge tube. Take care not to disturb the cell layer or transfer any cells.
6. Inspect plasma for turbidity. Turbid samples should be discarded and centrifuged and
aspirated again to remove remaining insoluble matter.
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Blood Serum Preparation

1. Draw the whole blood into centrifuge tubes containing no anticoagulant. Draw
approximately 2.5 times the volume needed for use.
2. Incubate in an upright position at room temperature (30-45 degrees) for approximately
60 mins to allow clotting.
3. Centrifuge at 1000-2000rcf. Do not use break to stop the centrifuge.
4. Carefully aspirate the supernatant (serum) and pool into a centrifuge tube, taking care
not to disturb the cell layer or transfer any cells. Use a clean pipette for each tube.
5. Inspect serum for turbidity. Turbid samples should be centrifuged and aspirated again
to remove insoluble matter.
OBSERVATIONS:
Three layers were distinctly seen in the centrifuge tube containing blood and anticoagulant. Serum was collected from the floating layer over the clotted blood.
RESULT :
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Ex.No:
Date :
ABO BLOOD GROUPING

AIM :
To identify the blood group by ABO blood grouping method.
BACKGROUND:
In 1900 Karl Lansteiner first reported the presence of two antigens namely A and
b on the surface of human red blood cells . Based on this discovery he divided human blood
cells in to three groups like A,B,O. in 1902, Decastell and Sturil recognize the existence of a
fourth group AB.
RBC of all ABO group possess a common antigen, the h antigen i.e the precursor for the
formation of A and B antigen.
ABO antigen consist of three allelic genes a, b and o. the a and b gene control the
synthe sis of specific enzymes responsible for the production of the basic antigenic
glycoprotein known as the H substance. The o gene is an amorph and cannot transform
the h substance. There is no known specific anti-O, hence only 4 phenotypes are
recognized(A,B,O&AB).
The Rh factor antibody was identified by Leving and Stetson.This is a complex
system coded by allelic genes at three closely related loci, alternative antigen Cc, Ee and
together with D or no D(recessive d). so person may inherit C DE from father and cde from
mother and have a genoty CDE/cde .Genotype cde/cde is Rh negative.
About 95% of individuals among Indian population and 85% individual of Caucasian
origin posses D(Rh) antigen on their erythrocytes. Human red blood cells are classified as Rh
+ve or Rh -ve depending upon the presence or absence of this antigen on their surface.
PRINCIPLE:
Human red blood cells possessing A and B antigen will agglutinate in the presence of
antibody directed towards the respective antigen.agglutination of RBC with anti-A
monoclonal .anti-B monoclonal a positive test result and indicates the presence of
corresponding antigen. Absence of agglutination of RBC with anti-a monoclonal, anti-b
monoclonal an a negative test result and indicates the absence of the corresponding antigen
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and termed as blood group O.Human RBC possessing B antigen are agglutinated by the
antibody directed against B(Rh) antigen.
REQUIREMENTS:
1. Pricking needle
2. Slides
3. Anti A,B,& D serum
4. Ethanol
PROCEDURE:
1.
Wash the slides and wipe with Ethanol.
2.
Wipe your finger with Ethanol.
3.
Prick it with needle(freash).
4.
Spot blood on the slide in three positions.
5.
Add anti-sera A,B&D on respective spot.
6.
Mix properly and cheak for agglutination.
RESULTS:
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Date :
WIDAL TEST
INTRODUCTION:
Enteric fever specific agglutinins (antibodies) are detected in patients after 15 days of
fever. BCG vaccinated patients serum may show elevated titre of all three H agglutinins.
Stained salmonella antigens are used to detect and identify specific antibodies in serum
samples from patients suffering from enteric fever.
PRINCIPLE:
Bacterial suspension which carry antigen will agglutinate on exposure to antibodies to
salmonella organisms.
SAMPLE:
Fresh serum is preferred. In case of any delay performing the test, serum should be
stored at 20 - 80 C.
STORAGE & STABILITY OF REAGENTS:
All reagents are ready to use and stable at 20 80 C till the expiry date.
REAGENTS:
1. Antigen suspension, S. typhi O.
2. Antigen suspension, S. typhi H.
3. Antigen suspension, S. paratyphi AH.
4. Antigen suspension, S. paratyphi BH.
5. Polyspecific positive cotrol
6. Glass Slides with 6 reaction circles and Mixing sticks.
Bring all reagents to Room Temperature before testing. Shake well antigens before actual use.
PROCEDURE:
1. Place one drop of positive control on one reaction circles of the slide.
2. Pipette one drop of Isotonic saline on the next reaction circle. (-ve Control)
3. Pipette one drop of the patient serum tobe tested onto the remaining four reaction
circles.
4. Add one drop of Widal TEST antigen suspension H to the first two reaction
circles.
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5. Add one drop each of O, H, AH and BH antigens to the remaining four

reaction circles.
6. Mix contents of each circle uniformly over the entire circle with separate mixing
sticks.
7. Rock the slide, gently back and forth and observe for agglutination macroscopically
within one minute.
Note :
Agglutination is a positive test result and if the positive reaction is observed with 20 ul
of test sample, it indicates presence of clinically significant levels of the corresponding
antibody in the patient serum.
No agglutination is a negative test result and indicates absence of clinically significant
levels of the corresponding antibody in the patient serum.
RESULT:
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Ex.No:
Date :
ASO LATEX TEST

INTRODUCTION:
It is a rapid latex agglutination test for the qualitative and semi-quantitative
determination of anti-streptolysin-O antibodies (ASO) in serum. In infections caused by haemolytic streptococci, streptolysin-O is one of the two hemolytic exotoxins liberated from
the bacteria that stimulates production of ASO antibodies in the human serum. The presence
and the level of these antibodies in a serum may reflect the nature and severity of infection.
The RapidTex ASO latex reagent is a stabilised buffered suspension of polystyrene latex
particles that have been coated with Streptolysin O.
When the latex reagent is mixed with a serum containing ASO, agglutination occurs.
The sensitivity of the latex reagent has been adjusted to yield agglutination when the level of
ASO is greater than 200 IU/ml, a level determined to be indicative of disease by
epidemiological and clinical studies.
Sera having titers of between 200 IU/ml to 3500 IU/ml will be reactive.
Sample Collection and Handling: Only fresh serum specimens should be used. Plasma must
not be used since fibrinogen may cause non-specific agglutination of the latex. It is preferable
to test samples on the same day as collected. Serum samples may be stored at 2-8o C for up to
48 hours prior to testing. If longer storage is necessary, sera should be stored frozen at 20C.
MATERIALS REQUIRED :
1.
ASO Antigen: A stabilized buffered suspension of polystyrene latex particles

coated with Streptolysin O and 0.1% sodium azide as preservative. Shake well
prior to use.
2.
ASO Positive Control: Human serum containing more than 200 IU/ml ASO and
0.1% sodium azide as preservative.
3.
ASO Negative Control: Human serum containing 0.1% sodium azide as

preservative.
4.
Sufficient disposable pipettes.
5.
Glass test slide.
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PROCEDURE:
1.
Bring all test reagents and samples to room temperature.
2.
Use a disposable pipette to draw up and place one free-falling drop of each
undiluted sample into its identified circle of the slide. Retain each pipette for
mixing in step 5.
3.
Deliver one free-falling drop of positive and negative control into its identified
circle.
4.
Mix the ASO latex reagent by gently shaking. Add one free-falling drop of
reagent to each control and sample.
5.
Using the flattened end of the appropriate plastic pipette as a stirrer (step 2),
thoroughly mix each sample with reagent within the full area of the circle.
6.
Discard the disposable pipette.
7.
Slowly rock the slide for exactly two (2) minutes and observe for agglutination
under a high intensity light.
8.
Record results.
9.
Re-wash glass slide for future use
RESULT:
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Ex.No:
Date :
PREGNANCY TEST
INTRODUCTION:
The hCG Urine Pregnancy Test Strip is a test kit for the determination of hCG (Human
Chorionic Gonadotropin) in urine specimens. This test kit is used to obtain a visual,
qualitative result for the early detection of pregnancy. Human chorionic gonadotropin (hCG)
is a glycoprotein hormone secreted by the developing placenta shortly after fertilization. The
appearance of hCG soon after conception and its subsequent rise in concentration during early
gestational growth make it an excellent marker for the early detection of pregnancy.
PROCEDURE:
1. To open the sealed pouch by tearing along the notch. Remove the test from the pouch.
Note: First morning urine usually contains the highest concentration of hCG and is
therefore the best sample when performing the urine test. However, randomly
collected urine specimens may be used.
2. To Hold the strip vertically, carefully dip it into the specimen (you may collect your
urine in a clean, dry container). Immerse the strip into the urine sample with the arrow
end pointing towards the urine. Do not immerse past the MAX Line (Marker Line).
Take the strip out after 10 seconds and lay the strip flat on a clean, dry, non-absorbent
surface. (Note: In rare instances when dye does not enter the result area, dip the tip of
the test strip in the urine as instructed above until the dye begins traveling across the
white result area).
3. Colored bands to appear. Depending on the concentration of hCG in the test specimen,
positive results may be observed in as little as 40 seconds. However, to confirm
negative results, the complete reaction time of 5 minutes is required. It is important
that the background is clear before the result is read. Do not read results after the
specified reaction time.
RESULT :
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Ex.No:
Date :
HAEMAGGLUTINATION TEST
AIM :
To determine the presence of a haemagglutination
INTRODUCTION:
All strains of Newcastle disease virus will agglutinate chicken red blood cells. This is
the result of the haemagglutinin part of the haemagglutinin/neuraminidase viral protein
binding to receptors on the membrane of red blood cells. The linking together of the red blood
cells by the viral particles results in clumping, this clumping is known as haemagglutination.
Haemagglutination is visible macroscopically and is the basis of haemagglutination
tests to detect the presence of viral particles. The test does not discriminate between viral
particles that are infectious and particles that are degraded and no longer able to infect cells.
Both can cause the agglutination of red blood cells.
Note that some other viruses and some bacteria will also agglutinate chicken red blood
cells. To demonstrate that the haemagglutinating agent is Newcastle disease virus, it is
necessary to use a specific Newcastle disease virus antiserum to inhibit the haemagglutinating
activity. Substances that agglutinate red blood cells are referred to as haemagglutinins.
MATERIALS:
1. Clean glass microscope slide or a clean white ceramic tile.
2. 10 percent suspension of washed chicken red blood cells.
3. Micropipette and tips, glass Pasteur pipette or a wire loop.
4. PBS.
5. Negative and positive control allantoic fluid samples.
6. Sample to be tested for the presence of Newcastle disease virus, for example
allantoic fluid.
PROCEDURE:
1. Place 4 separate drops of 10 percent chicken red blood cells onto a glass slide or a
white tile.
2. To each drop of blood, add one drop of the control and test samples as follows. Use
separate tips, pipettes or a flamed loop to dispense each sample.
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Drop 1 PBS
Drop 2 Negative control allantoic fluid (no haemagglutinin)
Drop 3 Positive control allantoic fluid (contains haemagglutinin)
Drop 4 Unknown sample to be tested
3. Mix by rotating the slide or tile for one minute.
4. Observe and record results. Compare results of the test samples with the control
samples.
RESULTS:
1. Agglutinated red blood cells in suspension have a clumped appearance distinct from
non-agglutinated red blood cells.
2. The red blood cells mixed with the positive control allantoic fluid will clump within
one minute.
3. The red blood cells mixed with the PBS and negative control allantoic fluid remain as
an even suspension and do not clump.
4. Judge the results of the test sample by comparison with the positive and negative
controls.
5. The PBS and negative allantoic fluid controls are used to detect clumping of the red
blood cells in the absence of virus. This is unlikely to occur. If it does occur, the test is
invalid.
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Ex.No :
Date :
IMMUNODIFFUSION
AIM:
To learn the technique of Ouchterlony double diffusion.
PRINCIPLE:
Immunodiffusion in gels encompasses a variety of techniques, which are useful
for the analysis of antigens and antibodies. An antigen reacts with a specific antibody
to form an antigen-antibody complex, the composition of which depends on the nature,
concentration and proportion of the initial reactants.
Immunodiffusion in gels are classified as single diffusion and double diffusion.
In ouchterlony double diffusion, both antigen and antibody are allowed to diffuse into
the gel. This assay is frequently used for comparing different antigen preparation. In
this test, different antigen preparations, each containing single antigenic species are
allowed to diffuse from separate wells against the antiserum. Depending on the
similarity between the antigens, different geometrical patterns are produced between
the antigen and antiserum wells. The patterns of lines form can be interpreted to
determine whether the antigens are same or different as illustrated below:
Pattern of identity: A
The antibodies in the antiserum react with both the antigen resulting in a smooth line
of precipitate. The antibodies cannot distinguish between the two antigens. i.e.) the two
antigens are immunologically identical.
Pattern of identity: B
In the pattern of partial identity, the antibodies in the antiserum react more with one
of the antigens than the other. The spur is thought to result from the determinants
present in one antigen but lacking in the other antigen.
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Pattern of identity: C
In the pattern of non-identity, none of the antibodies in the antiserum react with
antigenic determinants that may be present in both the antigen ie) the two antigens are
immunologically unrelated as far as that antiserum is concerned.
MATERIALS REQUIRED:
1. Agarose
2. 10X assay buffer
3. Antiserum(A, B, C)
4. Test antigens
5. Glass plate
6. Gel punch with syringe
7. Template
8. Assay buffer: PBS
PROCEDURE:
1. 25 ml of 1.2% Agarose (0.3g/25ml) was prepared in 1X assay buffer and
Agarose was dissolved completely by boiling.
2. The solutions were cooled at 50-60c and 4ml/plate was poured onto 5 grease
free glass plates placed on a horizontal surface. The gel was allowed to set for
30minutes.
3. Wells were punched by keeping the glass plate on the template.
4. The wells were filled with 10 l each of the antiserum and the corresponding
antigens.
5. The glass plates were kept in a moist chamber overnight at 37c.
6. After incubation, opaque precipitin lines between the antigen and antisera wells
were observed.
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RESULT:
1. Reaction of identity:
This occurs between identical antigenic determinants. The line of precipitation
is given as a continuous arc.
2. Reaction of non-identity:
When they do not contain any common antigenic determinant the two lines are
found independently and were without any interaction.
3. Reaction of partial-identity:
This has 2 components:
i. Those antigenic determinants which are common to both give a continuous line
of identity.
ii. The unique determinant recognition is one of the antigen, in addition a line of
non-identity, so that a spur is formed.
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Ex.No:
Date :
COUNTER CURRENT IMMUNO ELECTROPHORESIS (CCIE)

AIM:
To check the antisera for the presence of antibody towards a specific antigen by
counter current immunoelectrophoresis
PRINCIPLE:
Counter current immunoelectrpphoresis is a rapid version of Ouchterlony Double
Diffusion (ODD) technique which can be performed within one hour. It is primarily a
qualitative test although from the thickness of the precipitation line relative measure of
quantity can be obtained.
The antigen is placed in the well at the cathode and antibody is placed at the anode.
During electrophoresis molecules placed in an electric field acquire a charge depending on
their pI. Hence they move towards the appropriate electrodes. The antigen if it is negatively
charged moved towards the anode.
Antibody immunoglobulin at pH 7.6 has a charge nearing zero. During electrophoresis
the agarose matrix absorbs hydroxyl ions on the surface resulting in a net increase in positive
ions at a distance from the matrix. These positive ions migrate towards a negative pole with
the solvent shield resulting in a net solvent flow called endo osmosis. Hence antibody
molecules which have no charge move towards cathode along the solvent shield due to this.
Thus the antigen and antibody travel towards each other and at a point where there is optimum
concentration of both, a line of precipitin (band) is formed
REQUIREMENTS:
Conical flask, measuring Cylinder, distilled water, tips, micropipette, antigen, test
antiserum (1,2,3), positive control gel puncture, agarose, electrophoresis buffer.
PROCEDURE:
1.
Prepare 10ml of 1.5% agarose (.15g in 10 ml of buffer) in 1X electrophoresis

buffer by adding agarose to the buffer and heating slowly to dissolve the
agarose completely.
2.
Wipe the glass slide thoroughly and make it grease free for even spreading of
agarose.
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3.
Mark the ends of the slide which will be towards positive and negative
electrodes during electrophoresis respectively.
4.
Place the slide on the labelled table top and quickly pour the agarose and let it
spread. It is allowed to solidify for 15 min without any disturbance.
5.
Place the gel plate on the template holder and fix the template for CCIEP.
Punch 3mm wells with the gel puncher corresponding to the marking on the
template (punch 4 pairs of wells for the experiment).
6.
Place the slide in the electrophoresis tank and fill the tank with 1X
electrophoresis buffer till buffer just covers the gel surface. Dont add excess
buffer.
7.
Add 10l of antigen in each of the 4 wells towards cathode (negative electrode)
and 10l of antisera (1, 2, and 3) and 10l of positive control antisera towards
anode (positive electrode).
8.
Connect the power cord to the electrophoresis power supply (50V) and allow
the electrophoresis to continue for about 45 min.
9.
Observe for precipitin line between the antigen and antisera well.
INFERENCE:
Precipitin line indicates the presence of antibody present in the test serum for the
respective antigen.
RESULT:
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Ex.No:
Date :
ROCKET IMMUNO ELECTROPHORESIS

AIM:
To determine the concentration of a given antigen
PRINCIPLE:
Rocket immunoelectrophoresis, also known as electroimmunodiffusion is a simple,
quick, reproducible method for determining the concentration of antigen in an unknown
sample. Various concentrations of antigen are loaded side by side in small circular wells along
the edge of the agarose gel which contains specific antibody.
On electrophoresis, the antigen begins to migrate towards the anode and interacts with
antibody molecule to form a soluble antigen0 antibody complex. However, as the samples are
electrophoresed further more antibody molecules are encountered that interact with antigen
and when equivalence is reached =, the antigen-antibody complex precipitates. This precipitin
line is seen in the form of rocket. Higher the amount of antigen loaded in the gel, farther the
antigen will travel through the gel. Hence with increasing concentrations of antigen, a series
of rockets of increasing heights are seen that is proportional to the amount of antigen in the
well.
Therefore, a direct measurement of the height of the rocket will reflect on the antibody
concentration. The standard graph of antigen concentration Vs peak height is then constructed
and from the peak height of the unknown sample, concentration of antigen is determined.
REQUIREMENTS:
Glassware: Conical flask, Measuring cylinder. Reagents : Alcohol, Distilled water.
Other Requirements : Micropipette, Tips, Moist chamber (box with wet cotton).
PROCEDURE:
1. Prepare 10ml of 1% agarose in 1X electrophoresis buffer by adding agarose to the
buffer and heating slowly to dissolve the agarose completely. Take care not to froth
the solution.
2. Allow the molten agarose to cool to 55oC.
3. Add 1ml of antiserum to 10ml of agarose solution. Mix gently. Ensure uniform
distribution of antiserum.
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4. Pour the mix onto the glass plate placed on a horizontal surface and allow it to
solidify.
5. Place the glass plate on the template holder provided and fix the RIEP template.
6. Punch 3mm wells with gel puncher towards one edge of the plate.
7. Place the glass plate in the electrophoresis tank and ensure that the wells are towards
the cathode.
8. Fill the tank with 1X electrophoresis buffer till buffer just covers the gel surface.
9. Connect the power cord to the electrophoretic power supply.
10. Add 10l of each given standard antigen and test antigen to the wells.
NOTE: Loading of the wells should be carried out quickly to minimize diffusion from
the well.
11. Electrophores the sample at 100 V till the rockets are visible or the dye front reaches
the edge. This severally takes 1 to 1and 1/2 hours.
12. Electrophoresis can be continued for an additional 15 min after the dye has run out
from the gel. This ensures better visibility of the precipitation peaks.
13. Stop electrophoresis and remove the glass plate from the electrophoresis tank.
14. Observe the precipitation peak or rocket formed against a dark background. NOTE: If
the rockets are still not clear then incubate the plates in a moist chamber at 2 oC for
one hour or overnight.
15. Measure the rocket height from the upper edge of the well to the tip of the rocket.
Record your observation.
16. Construct a standard graph by plotting height of the peak along Y-axis against the
concentration of antigen on X-axis on a semi log Graph sheet (Y-axis linear scale and
X-axis log scale).
17. Determine the concentration of the antigen in a test sample by reading the
concentration against the rocket height from the standard graph.
RESULT:
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Ex.No:
Date :
DOT ELISA
AIM:
To perform sandwich dot ELISA test for the detection of antigen.
PRINCIPLE:
Dot ELISA is an extensively used immunological tool in research , as well as in
analytical and diagnostic laboratory. In this the antigen is sandwiched directly between the
antibodies which react with two different epitopes on the same antigen. Here one of the
antibodies is immobilized on to a solid support and the second antibody is linked to an
enzyme. Antigen in the test sample first reacts with the immobilized antibody and then with
the second enzyme linked antibody. Among the enzyme linked antibody bound this assay by
incubating the strip with an appropriate chromogenic substrate which is converted into a
coloured insoluble product. The latter precipitates on the strip in the area of enzyme activity
hence the name dot ELISA . The enzyme activity is indicated by the intensity of the spot
which is directly proportional to the antigen concentration.
MATERIALS REQUIREMENTS:
1. 1X assay buffer
2. Test serum sample
3. Dot ELISA strip
4. Antibody - HRP
5. Substrates TMB H2O2
PROCEDURE :
1.
In a vial take 1ml of 1X assay buffer and 50l of test serum sample. Mix thoroughly
and insert a dot ELISA strip.
2.
Allow the reaction to occur at room temperature for 20 minutes .
3.
Wash the strip by dripping it in 1ml of 1X assay buffer for about 5 minutes. Repeat it
thrice and replace the buffer each time.
5. Take 1ml of 1X assay buffer in a fresh vial and add 10l antibody HRP conjugate.
Mix thoroughly .
Namakkal
23
6. Dip the strip and allow the reaction to take place for 20 minutes.
7. Wash the strip by dipping it in 1ml of 1X assay buffer for about 5 minutes. Repeat it
thrice and replace the buffer each time.
8. In a fresh vial take 0.1ml of 10X TMB or H2O2 and 0.9ml of distilled water.
9. Dip the strip in the substrate solution .
10. Observe the strip after 10 to 20 minutes for the appearance of blue colour.
11. Rinse the strip with distilled water to stop the reaction.
INFERENCE:
Colored dot indicates the presence of specific antigen in the given sera.
RESULT:
Namakkal
24
Ex.No:
Date :
PREPARATION OF COMPETENT CELLS
Aim
Preparation of TOP10 competent cells by calcium chloride method
Principle
Competence is the ability of a cell to take up extra cellular DNA from its environment.
Competency can be artificially induced by treating the cells with CaCl2 prior to adding DNA.
The calcium destabilizes the cell membrane and adheres to the cell surface favoring the
formation of the pores for the entry of DNA.
Materials
LB plates with TOP10 cells
LB broth and plates
0.1M CaCl2
1.5ml centrifuge tubes
Spectrophotometer
Procedure
A single colony of TOP10 cells were inoculated inyo 2 ml of LB medium and incubated
overnight at 37C and at 150-200 rpm.
About 0.4 ml of the overnight culture was used to inoculate 40 ml of LB medium and
incubated at 37C at 150-200 rpm until the OD600 reaches 0.4 to 0.5.
About 1.5 ml of cell culture was transferred to centrifuge tubes and the cells were pelleted
down at 6000 rpm for 5 min at 4 oC.
The pellet was resuspended in 1ml of ice cold 0.1 M CaCl2 and incubated in ice for 30 min.
The cells were again pelleted down at 6000 rpm for 5 min at 4oC.
The pellet was dissolved in 120l of 0.1 M CaCl2 and 80l of 50% glycerol.
The check the viability a loop full of the above suspension were streaked onto a LB plates
and incubated at 37C for overnight
The remaining cell suspension were immediately transferred to -80C for future use
Observation
The colonies were seen on the plates
Inference
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25
The colonies growth infer the competent cells were viable and with out contamination
Ex.No:
Date :
TRANSFORMATION
Aim
Transforamtion of TOP10 cells with the pUC18-DNA ligated product
Principle
Changing the genotype of a cell or organism by transferring foreign DNA is called
transformation. The transferred DNA may be maintained as extra-chromosomal elements or
integrated into the genome. In this experiment, ampicillin susceptible genotype of E.coli strain
TOP10 are changed to ampicillin resistant genotype by transferring pUC18 plasmid that
carries a gene for ampicillin resistance and the selection in done in a selection medium with
ampicillin.
pUC18 vectors have a short segment of E. coli DNA, which contains the regulatory and
coding sequences of Lac Z gene that codes for -galactosidase enzyme. Isopropyl
thiogalactoside (IPTG) is an inducer of Lac Z gene expression. -galactosidase reacts with
the chromogenic substrate 5-bromo-4-chloro- -D-Galactoside (X-gal) and yields a blue
colored product. A multiple cloning site (MCS) is engineered inside the coding region of the
Lac Z gene. The MCS as such does not disrupt the reading frame and results only in insertion
of a few amino acids in the amino terminal fragment of the -galactosidase. Therefore, the
colonies appear blue in color in the presence of IPTG and X-gal. However, when a insert is
cloned in the MCS, that becomes a harmful insertion to the functional properties of galactosidase and it can no longer react with X-gal, and therefore, the colonies appear white in
color. This is a simple visual color test that can be used to screen thousands of colonies to
identify the presence of recombinant plasmids.
Materials
Competent TOP10 cells (Cells prepared from EXP 5)
Foreign DNA (Ligated product from EXP4)
LB medium
LB plates with 100mg/L Amp
42C water bath
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26
Isopropyl thiogalactoside (IPTG) 100mm, Sterilize by filtration.

5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) 20mg/ml dissolved in
DMSO.
Procedure
TOP10 competent cells were taken out from -80C freezer and thawed in the ice.
About 5ul of the ligated product were mixed with the competent cells and incubated in ice
for 30 min
The mixture was then subjected to heat shock at 42oC for 45 seconds.
About 500ul of LB broth was added in a sterile condition and incubated under shaking
conditions at 37C for 1 hr
Meanwhile, 40l of IPTG and 40l of X-gal were plated on top of the LB AMP plates, and
the plates were dried
About 100ul of the inoculum were plated on LB AMP plates containing and IPTG / X-gal,
incubated overnight at 37oC for 16 hrs.
About 100ul of untransformed TOP10 cells were plated on a LB AMP plates containing and
IPTG / X-gal as a negative control
Observation
Blue white colonies were seen on incubated plates.
Inference
Blue colonies indicate the self ligated product and the white colonies indicate the
recombined products.
Namakkal
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Ex.No :
Date :
AGAROSE GEL ELECTROPHORESIS
AIM:
DNA check run by Agarose Gel Electrophoresis
PRINCIPLE:
Agarose gel electrophoresis separates DNA fragments according to their size. An
electric current is used to move the DNA molecules across an agarose gel, which is a
polysaccharide matrix that functions as a sieve to help "catch" the molecules as they are
transported by the electric current.
The phosphate molecules that make up the backbone of DNA molecules have a high
negative charge. When DNA is placed on a field with an electric current, these negatively
charged DNA molecules migrate toward the positive end of the field, which in this case is an
agarose gel immersed in a buffer bath. The agarose gel is a cross-linked matrix i.e., a threedimensional mesh or screen. The DNA molecules are pulled to the positive end by the current,
but they encounter resistance from this agarose mesh. The smaller molecules are able to
navigate the mesh faster than the larger ones. This is how agarose electrophoresis separates
different DNA molecules according to their size. The gel is stained with ethidium bromide so
as to visualize these DNA molecules resolved into bands along the gel.Ethidium bromide is an
intercalcating dye, which intercalate between the bases that are stacked in the center of the
DNA helix. One ethidium bromide molecule binds to one base. As each dye molecule binds to
the bases the helix is unwound to accommodate the stain from the dye. Closed circular DNA
is constrained and cannot withstand as much twisting strain as can linear DNA, so circular
DNA cannot bind as much dye as can linear DNA.
Unknown DNA samples are typically run on the same gel with a "ladder." A ladder is
a sample of DNA where the sizes of the bands are known. Unknown fragments are compared
with the ladder fragments (size known) to determine the approximate size of the unknown
DNA bands.
Approximately 10ng is visible in a single band on a horizontal agarose gel.
MATERIALS:
1. Agarose
2. TBE buffer
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3. Gel casting tray, comb, power pack

4. Sample DNA
5. Loading dye
6. Sterile microtips
7. EtBr staining solution
8. UV transilluminator or Gel Documentation System
PROCEDURE :
For casting gel, agarose powder was mixed with electrophoresis buffer (TBE) to the
desired concentration, then heated in a microwave oven until completely melted. After
cooling the solution to about 600C, it was poured into a casting tray containing a comb and
allowed to solidify at room temperature for nearly 45 min.
After the gel has solidified, the comb was removed, using care not to rip the bottom of
the wells. The gel, still in its plastic tray, was inserted horizontally into the electrophoresis
chamber and just immersed with buffer (TBE). DNA samples mixed with loading buffer were
then pipeted into the sample wells, the lid and power leads were placed on the apparatus, and
a current was applied. The current flow was confirmed by observing bubbles coming off the
electrodes. DNA will migrate towards the positive electrode, which is usually colored red.
The distance DNA has migrated in the gel can be judged by visually monitoring migration of
the tracking dyes. Bromophenol blue and xylene cyanol dyes migrate through agarose gels at
roughly the same rate as double-stranded DNA fragments of 300 and 4000 bp, respectively.
When adequate migration (2/3 of the gel) has occurred, DNA fragments were visualized by
staining with ethidium bromide. This fluorescent dye intercalates between bases of DNA and
RNA. It was often incorporated into the gel so that staining occurs during electrophoresis, but
the gel can also be stained after electrophoresis by soaking in a dilute solution of ethidium
bromide. To visualize DNA or RNA, the gel was placed on a ultraviolet transilluminator. Be
aware that DNA will diffuse within the gel over time, and examination or photography should
take place shortly after cessation of electrophoresis.
Preparation of 0.7% Agarose gel:
0.35 g agarose was weighed, 50 ml 1X TBE was added to it and agarose was melted in
a microwave oven for 2-3 min and Cooled down to about 45 to 500 C (bearable warmth) and
poured into the gel platform with the comb in position.
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Running gel:
After solidification of the gel (approx. 45 min), place the gel in a gel tank with 1 X
TBE buffer. Buffer should be filled to the surface of the gel. Load the samples in the well and
run the gel at 60 V till the blue dye runs to the end.
Staining the gel:
The staining solution was prepared by adding 10 l of 10 mg/ml stock of Ethidium
bromide in 100 ml of dd water. The gel was placed in staining solution for 30 min and view
the gel in UV transilluminator.
Gel loading dye: 10X stock (10 ml)
Bromophenol blue 0.25%
25%
Ficoll
25 mg of bromophenol blue was weighed and dissolved in 7 ml of sterile dd water, in

a screw cap tube. 2.5 g of ficoll was added and dissolved completely (keep the tube in a
shaker, overnight). Measure the volume using a pipette and made upto 10 ml using sdd water.
Stored at 40 C.
10X TBE (pH 8.2): 1000 ml
Tris
107.78 g
EDTA
8.41 g
Boric acid
55 g
Dissolved in 600 ml of dd water( First allow the Tris to dissolve in water, then add
EDTA). The volume was made upto to one liter and autoclave it. (Check and confirm the pH
is about 8.2)
Ethidium Bromide Stock:
Stock 10 mg/ml working concentration 1 g/ml.
NOTES:
Fragments of linear DNA migrate through agarose gels with a mobility that is
inversely proportional to the log10 of their molecular weight. In other words, if you plot the
distance from the well that DNA fragments have migrated against the log10 of either their
molecular weights or number of base pairs, a roughly straight line will appear.
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1. Circular forms of DNA migrate in agarose distinctly differently from linear DNAs of
the same mass. Typically, uncut plasmids will appear to migrate more rapidly than the
same plasmid when linearized. Additionally, most preparations of uncut plasmid
contain at least two topologically different forms of DNA, corresponding to
supercoiled forms and nicked circles. The image to the right shows an ethidiumstained gel with uncut plasmid in the left lane and the same plasmid linearized at a
single site in the right lane.
2. Several additional factors have important effects on the mobility of DNA fragments in
agarose gels, and can be used to your advantage in optimizing separation of DNA
fragments. Chief among these factors are:
3. Agarose Concentration: By using gels with different concentrations of agarose, one
can resolve different sizes of DNA fragments. Higher concentrations of agarose
facilite separation of small DNAs, while low agarose concentrations allow resolution
of larger DNA
4. The image in the right shows migration of a set of DNA fragments
in three
concentrations of agarose, all of which were in the same gel tray and electrophoresed
at the same voltage and for identical times. Notice how the larger fragments are much
better resolved in the 0.7% gel, while the small fragments separated best in 1.5%
agarose. The 1000 bp fragment is indicated in each lane.
5. Voltage: As the voltage applied to a gel is increased, larger fragments migrate
proportionally faster those small fragments. For that reason, the best resolution of
fragments larger than about 2 kb is attained by applying no more than 5 volts per cm to
the gel (the cm value is the distance between the two electrodes, not the length of the
gel).
6. Electrophoresis Buffer: Several different buffers have been recommended for
electrophoresis of DNA. The most commonly used for duplex DNA are TAE (Trisacetate-EDTA) and TBE (Tris-borate-EDTA). DNA fragments will migrate at
somewhat different rates in these two buffers due to differences in ionic strength.
Buffers not only establish a pH, but provide ions to support conductivity. If you
mistakenly use water instead of buffer, there will be essentially no migration of DNA
Namakkal
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in the gel! Conversely, if you use concentrated buffer (e.g. a 10X stock solution),
enough heat may be generated in the gel to melt it.
7. Effects of Ethidium Bromide: Ethidium bromide is a fluorescent dye that intercalates
between bases of nucleic acids and allows very convenient detection of DNA
fragments in gels, as shown by all the images on this page. As described above, it can
be incorporated into agarose gels, or added to samples of DNA before loading to
enable visualization of the fragments within the gel. As might be expected, binding of
ethidium bromide to DNA alters its mass and rigidity, and therefore its mobility.
RESULT:
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Ex.No:
Date :
ISOLATION OF GENOMIC DNA FROM E. COLI

AIM:
To isolate the genomic DNA from E .coli DH5 cells.
PRINCIPLE:
The isolation and purification of DNA from cells is one of the most common
procedures in contemporary molecular biology and embodies a transition from cell biology to
the molecular biology (from in vivo to in vitro). The isolation of DNA from bacteria is a
relatively simple process. The organism to be used should be grown in a favorable medium at
an optimal temperature, and should be harvested in late log to early stationary phase for
maximum yield.
The genomic DNA isolation needs to separate total DNA from RNA, protein, lipid,
etc. Initially the cell membranes must be disrupted in order to release the DNA in the
extraction buffer. SDS (sodium dodecyl sulphate) is used to disrupt the cell membrane. Once
cell is disrupted, the endogenous nucleases tend to cause extensive hydrolysis. Nucleases
apparently present on human fingertips are notorious for causing spurious degradation of
nucleic acids during purification. DNA can be protected from endogenous nucleases by
chelating Mg2++ ions using EDTA. Mg2++ ion is considered as a necessary cofactor for
action of most of the nucleases. Nucleoprotein interactions are disrupted with SDS, phenol or
proteinase K. Proteinase enzyme is used to degrade the proteins in the disrupted cell soup.
Phenol and chloroform are used to denature and separate proteins from DNA. Chloroform is
also a protein denaturant, which stabilizes the rather unstable boundary between an aqueous
phase and pure phenol layer. The denatured proteins form a layer at the interface between the
aqueous and the organic phases which are removed by centrifugation. DNA released from
disrupted cells is precipitated by cold absolute ethanol or isopropanol.
MATERIALS REQUIRED:
1. LB Broth
2. E. coli DH5 cells
3. Reagents
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4. TE buffer (pH 8.0)

5. 10% SDS
6. Proteinase K
7. Phenol-chloroform mixture
8. 5M Sodium Acetate (pH 5.2)
9. Isopropanol
10. 70% ethanol
PREPARATION OF REAGENTS:
1. TE BUFFER (pH 8.0): 10 mm Tris HCl (pH 8.0), 1 mm EDTA (pH 8.0)
2. 10% SDS: Dissolve 10 g of SDS in 100 ml autoclaved distilled water.
3. PROTEINASE K: Dissolve 10 mg of Proteinase K in 1 ml autoclaved distilled water.
4. PHENOL CHLOROFORM MIXTURE: The pH is very important. For RNA
purification, the pH is kept around pH 4, which retains RNA in the aqueous phase
preferentially. For DNA purification, the pH is usually 7 to 8, at which point all
nucleic acids are found in the aqueous phase. Mix equal volume of phenol with
chloroform. Keep the mixture on ice and add 20 ml TE buffer, extract by shaking for
15 minutes. Remove the dust on the surface layer using a pipette. Repeat 4-5 times.
Add 30-40 ml of TE buffer and store it on ice.
5. 5M SODIUM ACETATE: Dissolve 41 g of sodium acetate in 100 ml distilled water
and adjust pH with dilute acetic acid (pH 5.2).
6. ISOPROPANOL
7. 70% ETHANOL
PROCEDURE:
1. 2 ml overnight culture is taken and the cells are harvested by centrifugation for 10
minutes
2. 875 l of TE buffer is added to the cell pellet and the cells are resuspended in the
buffer by gentle mixing.
3. 100 l of 10% SDS and 5 l of Proteinase K are added to the cells.
4. The above mixture is mixed well and incubated at 37 C for an hour in an incubator.
Namakkal
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5. 1 ml of phenol-chloroform mixture is added to the contents, mixed well by inverting

and incubated at room temperature for 5 minutes.
6. The contents are centrifuged at 10,000 rpm for 10 minutes at 4 C.
7. The highly viscous jelly like supernatant is collected using cut tips and is transferred to
a fresh tube.
8. The process is repeated once again with phenol-chloroform mixture and the
supernatant is collected in a fresh tube.
9. 100 l of 5M sodium acetate is added to the contents and is mixed gently.
10. 2 ml of isopropanol is added and mixed gently by inversion till white strands of DNA
precipitates out.
11. The contents are centrifuged at 5,000 rpm for 10 minutes. The Supernatant is removed
and 1ml 70% ethanol is added.
12. The above contents are centrifuged at 5,000 rpm for 10 minutes.
13. After air drying for 5 minutes 200 l of TE buffer or distilled water is added.
14. 10 l of DNA sample is taken and is diluted to 1 or 2 ml with distilled water.
15. The concentration of DNA is determined using a spectrophotometer at 260/280 nm.
16. The remaining samples are stored for further experiments.
PRECAUTIONS:
Cut tips should be used so that the DNA is not subjected to mechanical disruption.
Depending on the source of DNA the incubation period of Proteinase K should extend.
The phenol chloroform extraction should be repeated depending on the source of DNA to
obtain pure DNA.
DNase free plastic wares and reagents should be used.
RESULTS :
Namakkal
35
Ex.No:
Date :
PLASMID DNA ISOLATION
Aim:
To isolate plasmid DNA from bacterial cells
Principle:
When bacteria are lysed under alkaline conditions both DNA and proteins are
precipitated. After the addition of acetate-containing neutralization buffer the large and less
supercoiled chromosomal DNA and proteins precipitate, but the small bacterial DNA
plasmids can renature and stay in solution.
In prokaryotes, plasmid is double stranded, circular, and is found in the cytoplasm.
The cell membranes must be disrupted in order to release the plasmid in the extraction buffer.
Solution 1 contains glucose, Tris, and EDTA. Glucose provides osmotic shock leading to the
disruption of cell membrane, Tris is a buffering agent used to maintain a constant pH8.
Plasmid can be protected from endogenous nucleases by chelating Mg2++ ions using EDTA.
Mg2++ ion is considered as a necessary cofactor for most nucleases. Solution II contains
NaOH and SDS and this alkaline solution is used to disrupt the cell membrane and NaOH also
denatures the DNA into single strands. Solution III contains acetic acid to neutralise the pH
and potassium acetate to precipitate the chromosomal DNA, proteins, along with the cellular
debris. Phenol /chloroform is used to denature and separate proteins from plasmid.
Chloroform is also a protein denaturant, which stabilizes the rather unstable boundary
between an aqueous phase and pure phenol layer. The denatured proteins form a layer at the
interface between the aqueous and the organic phases which are removed by centrifugation.
Once the plasmid DNA is released, it must be precipitated in alcohol. The plasmid DNA in
the aqueous phase is precipitated with cold (0oC) ethanol or isopropanol. The precipitate is
usually redissolved in buffer and treated with phenol or organic solvent to remove the last
traces of protein, followed by precipitation with cold ethanol.
MATERIALS REQUIRED:
1. Luria Broth
2. Bacterial cells containing plasmid
3. Reagents
4. TE buffer(pH 8.0)
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36
5. Solution I
6. Solution II
7. Solution III
8. Phenol-chloroform mixture
9. Isopropanol
PREPARATION OF REAGENTS:
1.
TE BUFFER (pH 8.0): 10 mm Tris HCl (pH 8.0) 1 mm EDTA (pH 8.0)
2.
Solution I: Lysis solution
3.
Solution II: Denaturing solution
3.
Solution III: Neutralizing solution
4.
PHENOL CHLOROFORM MIXTURE: Mix equal volume of phenol with
chloroform. Keep the mixture on ice and add 20 ml TE buffer, extract by shaking for 15
minutes. Remove the dust on the surface layer using a pipette. Repeat 4-5 times. Add 30-40
ml of TE buffer and store it in dark.
5.
ISOPROPANOL
PROCEDURE:
1. Take 2 ml overnight culture and harvest cells by centrifugation for 5 minutes. Discard
the supernatant carefully.
2. Add 100 l of solution I to the cell pellet and resuspend the cells by gentle mixing.
3. Incubate the above mixture at room temperature for 5 minutes.
4. Add 200 l of solution II to the mixture and mix by inverting the tubes for 5 minutes.
5. Incubate for 5-10 minutes at room temperature.
6. Add 500l of ice cold solution III to the mixture and mix by inverting the tube.
7. Incubate on ice for 10 minutes.
8. Centrifuge at 10,000 rpm for 5 minutes. Transfer the supernatant into fresh tube.
9. Add 400 l of phenol-chloroform mixture to the contents, mix well by inverting and
incubate them at room temperature for 5 minutes.
10. Centrifuge at 10000 rpm for 5 minutes.

11. Collect the supernatant (viscous) using cut tips and transfer to a fresh tube.
Namakkal
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12. Add 0.8 ml of isopropanol and mix gently by inversion. Incubate for 30 min at room
temperature.
13. Centrifuge the contents at 10,000 rpm for 10 minutes.
14. Discard the supernatant after centrifugation.
15. After air drying for 5 minutes, add 100 l of TE buffer or autoclaved distilled water to
the pellet to resuspend the plasmid DNA. The contaminated salt in the DNA pellet can
be removed with 70% ethanol washing.
16. Take 10 l of plasmid sample and dilute to 1 ml with distilled water for spectrometric
analysis.
17. The concentration of plasmid is determined using a spectrophotometer at 260/280 nm.
18. An aliquot of plasmid DNA is used for agarose electrophoresis for quantitative and
qualitative analyses.
RESULTS :
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Ex.No:
Date :
RESTRICTION ENZYME DIGESTION

AIM:
To digest the pUC18 DNA with BamH1 enzyme
PRINCIPLE:
Restriction endonucleases are the class of enzymes that are used to cleave DNA at
specific sites called Restriction sites. Every restriction enzyme has a specific restriction site at
which it cuts a DNA molecule. For example restriction sequence for BamHI is GGATCC
(type II restriction enzyme. The most abundantly used restriction enzymes are type II
restriction enzymes which cleave at specific restriction site only. These endonucleases
function adequately at pH 7.4 but different enzymes vary in their requirements for ionic
strength usually provided by sodium chloride and magnesium chloride. It is also advisable to
add a reducing agent such as dithiothreitol (DTT) which stabilizes the enzymes and prevents
their inactivation. Any variation in the concentration of Na or Mg can lead to changes in
specificity of enzyme so that it can cleave at additional or non standard restriction sequences.
The phosphodiester bond is cleaved between specific bases, one on each DNA strand, no
matter the source of the DNA. The restriction endonucleases produce either sticky or blunt
ends upon cleavage. Also based on the number of sequences identified for cleavage they can
be tetracutter (4), hexacutter (6) or octacutter (8).
MATERIALS REQUIRED:
1. pUC18 DNA
2. BamH1 enzyme 10X buffer
3. 1Kb Ladder Sterile water Agarose
4. 6X loading dye
5. 1.5 ml Sterile Vials Ethidium Bromide 1X TAE buffer
PROCEDURE:
1. Take 1.5 g of PUC18 DNA (10 ul) in a fresh eppendorf.
2. To this, add 11.5 l of sterile water followed by 5 l of 10X buffer.
3. Add 1.5 l of BamH1 enzyme (1 units) and incubate the mixture at 37C for 2 hrs.
Namakkal
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4. Prepare 0. 7% agarose gel and load the samples including 1 Kb DNA ladder,
undigested pUC18 DNA and BamH1 digested PUC18 DNA.
5. Run the gel at 100 V for 1 hr.
6. Visualize the gel under UV illuminator.
7. 10ul of the sample and 2ul of the dye were mixed Laod 10ul of this in to the gel
PROCEDURE :
Materials
Qty
PUC18 DNA
10 l
Deionized water
11l
10X buffer
3 l
BamH1
1 l
Total
25 l
(Incubate at 37 C for 12 hrs)

RESULTS:
Namakkal
40
Ex.No:
Date :
DNA LIGATION
AIM:
To perform the ligation of linearized T-vector with DNA fragment or the ligation of
any restriction enzyme digested DNA fragments using T4 DNA ligase.
PRINCIPLE:
The basic strategy in molecular cloning is to insert a DNA fragment of interest (a
segment of DNA) into a DNA molecule (called a vector) that is capable of independent
replication in a host cell. The result is a recombinant molecule composed of the DNA insert
linked to vector DNA sequences. Construction of these recombinant DNA molecules is
dependent on the ability to covalently seal single stranded nicks in DNA. This process is
accomplished both in vivo and in vitro by the enzyme DNA ligase. DNA ligation is the
process of joining together two DNA molecules ends (either from the same or different
molecules). The enzyme that joins the DNA fragments is called DNA ligases. The DNA
ligase seals the nicks in DNA by formation of phosphodiester bond between adjacent 3
hydroxyl and 5 phosphate termini. The enzyme extensively used in joining DNA fragments is
T4 DNA ligase. The ligase joins both cohesive end as well as blunt ended DNA. It is a single
polypeptide with a M.W of 68,000 Dalton requiring ATP as energy source. The maximal
activity pH range is 7.5-8.0. The enzyme exhibits 40% of its activity at pH 6.9 and 65% at pH
8.3. The DNA fragment (PCR product) has an extra A at 3 end so that it can
complementally bind to the T at the 5 end of the T- vector.
MATERIALS REQUIRED:
1. Restriction digested T-vector and PCR product (DNA) T4 DNA ligase
2. Ligation buffer
3. Nuclease free distilled water (autoclaved) Agarose
4. Gel loading dye Ethidium Bromide Micropipettes
5. Micro tips Microfuge
6. 50x TAE buffer
7. Electrophoresis unit and power supply Microwave oven/heater
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8. UV transilluminator/ Gel Doc
PROCEDURE:
1. Three separate vials are taken and are labelled as reaction, +ve control and ve control.
2. 1.5l of PCR product of DNA fragment is added to reaction and ve control vials
only.
3. 1 l of 10X Ligation buffer is added to each of the three vials.
4. 1 l of T4 DNA Ligase (1 U) is added to reaction and +ve control vials only.
6.5 l, 8 l, 7.5 l of water is added to reaction, +ve control and ve control vials,
respectively.
5. The total volume in each of the vials is 10 l. Incubate for 1 hr at 37 C.
6. The prepared mixtures can be analyzed in bacterial transformation in bacterial cells or
they can be analyzed onto agarose gel.
Materials
Qty
Double digested DNA (HIND III, BAM HI
2l
10X Ligation buffer
1l
T4 DNA Ligase
1l
Water
6l
Incubate for 1 hr at 37 C.
RESULTS:
Namakkal
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Ex.No:
Date :
Biological Databases with Reference to Expasy and NCBI

AIM:
To view and use the various biological databases available on the World Wide Web.
INTRODUCTION:
Biological data are highly complex and interrelated. Vast amount of biological
information needs to be stored organized and indexed so that the information can be
retrieved and used. There are five major types of databases namely nucleotide databases,
protein databases, protein structure databases, metabolic pathway databases and the
bibliographic databases.
PROCEDURE:
1.
Open your web browser and type the web address of the required database.
2.
Explore the database and analyze the various information available in the database.
3.
Use the tools provided by the databases.
4.
Save the output into a separate folder.
A. Expasy (Expert Protein analysis system):

Expasy is a proteomic server maintained by Swiss Institute of Bioinformatics for
providing information on protein structures. The Database works in collaboration with
European bioinformatics institute. Expasy is updated frequently with sequence information
and tools for analyzing protein sequences.
Introduction:
Expasy can be reached by typing the URL www.expasy.org which is maintained by
the Swiss institute of Bioinformatics. The website has a navigation column on the left side of
the window, where the whole web server is categorized into various fields like proteomics,
genomics, phylogeny , systems biology etc to create a better user experience. Each category is
divided into two sections, Databases and tools. Since Expasy serves as the warehouse of many
other databases and tools, all the available databases and tools available are characterized
under these categories.
Search Tool and categories:
The home page is loaded with a query search tool where the user can search for
Namakkal
43
biological information inside Expasy. A drop down menu is also provided in order to narrow
down the search. The results will feature no of hits for the query with respect to each and
every database based on which the user can direct himself to the location of information.
The Categories include
Proteomics
Protein sequences and identification(Databases and tools involved in it )
Post translational modification
Protein Structure
Protein Protein interaction
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Lab in Immunology, rDNA

Technology and Bioinformatics
Manual
Genomics
Structural Bioinformatics
System Biology
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Phylogeny/evolution
Services :
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46
B. NCBI ( National centre for Biotechnological information) :

NCBI is one of the leading online resources known for providing Biological sequence
information. NCBI is maintained by two organizations in US ,National Library of Medicine (
NLM) and National Institute of science ( NIH). As a national resource for molecular biology
information, NCBI's mission is to develop new information technologies to aid in the
understanding of fundamental molecular and genetic processes that control health and disease.
More specifically, the NCBI has been charged with creating automated systems for storing and
analyzing knowledge about molecular biology, biochemistry, and genetics.
NCBI is connected to various other sequence databases in order to be more efficient in
answering sequence queries. The user queries and sequence information are delivered through
NCBIs search tool called the entrez.
Home Page:
NCBI has a simplified homepage from where the user can navigate to different resources.
The left side pane of the Homepage has a site map followed by different categories which
narrows down the possibility of finding the right sequence. On the right side , you can see the list
of popular resources which is very useful for first time users.
GENBANK
The GenBank sequence database is an open access, annotated collection of all publicly
available nucleotide sequences and their protein translations. This database is produced and
maintained by the National Center for Biotechnology Information (NCBI) as part of the
International Nucleotide Sequence Database Collaboration (INSDC). The National Center for
Biotechnology Information is a part of the National Institutes of Health in the United States.
GenBank and its collaborators receive sequences produced in laboratories throughout the world
from more than 100,000 distinct organisms. In more than 20 years since its establishment,
GenBank has become the most important and most influential database for research in almost all
biological fields, whose data were accessed and cited by millions of researchers around the
world. GenBank continues to grow at an exponential rate, doubling every 18 months.
Entrez:
The NCBI database accepts queries and delivers data via a custom made search engine called
Entrez. The Home page of NCBI has a search box which directs the user to entrez. Entrez is
internally connected to various biological databases which increases the probability of getting the
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correct information
BLAST:
BLAST stands for Basic Local Alignment Search Tool.BLAST is a tools that is used to find the
seqyuences homologous to a particular sequence.BLAST compares all the sequences in the
database with the one that is searched for and provides many hits which are usually arranged in
the increasing order of the scored obtained
BLAST is available at the URLhttp://blast.ncbi.nlm.nih.gov/
BLAST uses PAM and BLOSUM matrices for scoring the alignment.
PubMed :
This is an online Bibliographic database which has a collection of the research papers, journals
and other bibliographic data. The Database is internally connected with other Bibliographic
databases like Medline, Biomedcentral etc.
Pubchem :
This contains data about the chemical compounds that are used for insillico analysis
Database of SNPs:
This database contains data about SNPs (Single Nucleotide polymorphism)
OMIM:
OMIM stand for Online Mendelian Inheritance in Man. This database contains information about
the genetical disorders. OMIM gives complete data on the diseases the genetical background
behind it and also the corresponding journal resources.
OMIA:
This database is similar to OMIM, but contains data about the diseases of all the other animals at
the genetic level except human.
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Lab in Immunology, rDNA Technology and Bioinformatics

Manual
The home page of NCBI can be seen as follows:
Output:
The file format of the particular protein keratin can be shown follows:
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Ex.No:
Date :
QUERIES BASED ON BIOLOGICAL DATABASES

Introduction:
Biological databases are libraries of life sciences information, collected from
scientific experiments, published literature, high-throughput experiment technology, and
computational analyses. They contain information from research areas including genomics,
proteomics, metabolomics, microarray gene expression, and phylogenetics. Information
contained in biological databases includes gene function, structure, localization (both cellular
and chromosomal), clinical effects of mutations as well as similarities of biological sequences
and structures.
Biological databases are an important tool in assisting scientists to understand and
explain a host of biological phenomena from the structure of biomolecules and their
interaction, to the whole metabolism of organisms and to understanding the evolution of
species. This knowledge helps facilitate the fight against diseases, assists in the development
of medications and in discovering basic relationships amongst species in the history of life.
Biological knowledge is distributed amongst many different general and specialized
databases. This sometimes makes it difficult to ensure the consistency of information.
Biological databases cross-reference other databases with accession numbers as one way of
linking their related knowledge together.
An important resource for finding biological databases is a special yearly issue of the
journal Nucleic Acids Research (NAR). The Database Issue of NAR is freely available, and
categorizes many of the publicly available online databases related to biology and
bioinformatics.
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Ex.No:
Date :
RETRIEVE THE GENE SEQUENCE IN FASTA FORMAT CORRESPONDING TO P00519
Aim:
To retrieve the gene sequence in FASTA format corresponding to P00519
Introduction:
A gene is a molecular unit of heredity of a living organism. It is a name given to some
stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a
function in the organism. Knowledge of gene sequences has become indispensable for basic
biological research, other research branches utilizing sequencing, and in numerous applied
fields such as
diagnostic, biotechnology, forensic biology and biological systematics.
In bioinformatics, FASTA format is a text-based format for representing either
nucleotide sequences or peptide sequences, in which nucleotides or amino acids are
represented using single-letter codes. The format also allows for sequence names and
comments to precede the sequences. The format originates from the FASTA software
package, but has now become a standard in the field of bioinformatics.
The simplicity of FASTA format makes it easy to manipulate and parse
sequences using text-processing tools and scripting languages
Method:
1. Open Uniprot Database www.uniprot.org
2. Enter the protein Id P00519 in search tab and click on Find
3. Click
on
displayed
the
on
the
protein
name
result
page.
ABL1_HUMAN
4. Obtain relevant information about protein and retrieve FASTA
format of its sequence by clicking on the FASTA tab at the right
corner.
Result and inference :
ABL1_HUMAN
P00519, A3KFJ3, Q13869, Q13870, Q16133, Q17R61, Q45F09
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Tyrosine-protein kinase ABL1

Organism: Homo sapiens
Function of Protein:
Non-receptor tyrosine-protein kinase that plays a role in many key processes linked to cell
growth and survival such as cytoskeleton remodeling in response to extracellular stimuli,
cell motility and adhesion, receptor endocytosis, autophagy, DNA damage response and
apoptosis. Coordinates actin remodeling through tyrosine phosphorylation of proteins
controlling cytoskeleton dynamics.
Catalytic Activity: ATP + a [protein]-L-tyrosine = ADP + a [protein]-L-tyrosine phosphate
Cofactor: Magnesium or manganese
Enzyme regulation: Stabilized in the inactive form by an association between the SH3
domain and the SH2-TK linker region, interactions of the N-terminal cap, and contributions
from an N-terminal myristoyl group and phospholipids.
Protein attributes
Sequence length
1130 AA.
Sequence status
Complete.
FASTA format of sequence:

>sp|P00519|ABL1_HUMAN Tyrosine-protein kinase ABL1 OS=Homo sapiens GN=ABL1 PE=1
SV=4
MLEICLKLVGCKSKKGLSSSSSCYLEEALQRPVASDFEPQGLSEAARWNSKENLLAGPSE
NDPNLFVALYDFVASGDNTLSITKGEKLRVLGYNHNGEWCEAQTKNGQGWVPSNYITP
VNSLEKHSWYHGPVSRNAAEYLLSSGINGSFLVRESESSPGQRSISLRYEGRVYHYRINT
ASDGKLYVSSESRFNTLAELVHHHSTVADGLITTLHYPAPKRNKPTVYGVSPNYDKWE
MERTDITMKHKLGGGQYGEVYEGVWKKYSLTVAVKTLKEDTMEVEEFLKEAAVMKEI
KHPNLVQLLGVCTREPPFYIITEFMTYGNLLDYLRECNRQEVNAVVLLYMATQISSAME
YLEKKNFIHRDLAARNCLVGENHLVKVADFGLSRLMTGDTYTAHAGAKFPIKWTAPES
LAYNKFSIKSDVWAFGVLLWEIATYGMSPYPGIDLSQVYELLEKDYRMERPEGCPEKVY
ELMRACWQWNPSDRPSFAEIHQAFETMFQESSISDEVEKELGKQGVRGAVSTLLQAPEL
PTKTRTSRRAAEHRDTTDVPEMPHSKGQGESDPLDHEPAVSPLLPRKERGPPEGGLNED
ERLLPKDKKTNLFSALIKKKKKTAPTPPKRSSSFREMDGQPERRGAGEEEGRDISNGALA
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FTPLDTADPAKSPKPSNGAGVPNGALRESGGSGFRSPHLWKKSSTLTSSRLATGEEEGG
GSSSKRFLRSCSASCVPHGAKDTEWRSVTLPRDLQSTGRQFDSSTFGGHKSEKPALPRKR
AGENRSDQVTRGTVTPPPRLVKKNEEAADEVFKDIMESSPGSSPPNLTPKPLRRQVTVAP
ASGLPHKEEAGKGSALGTPAAAEPVTPTSKAGSGAPGGTSKGPAEESRVRRHKHSSESP
GRDKGKLSRLKPAPPPPPAASAGKAGGKPSQSPSQEAAGEAVLGAKTKATSLVDAVNS
DAAKPSQPGEGLKKPVLPATPKPQSAKPSGTPISPAPVPSTLPSASSALAGDQPSSTAFIPL
ISTRVSLRKTRQPPERIASGAITKGVVLDSTEALCLAISRNSEQMASHSAVLEAGKNLYTF
CVSYVDSIQQMRNKFAFREAINKLENNLRELQICPATAGSGPAATQDFSKLLSSVKEISDI
VQR
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Ex.No:
Date:
SEQUENCE SIMILARITY SEARCHING USING BLAST

Introduction
Basic local alignment search tool (BLAST) is a sequence similarity search program.
The National Center for Biotechnology Information (NCBI) maintains a BLAST server with
a home page at http://www.ncbi.nlm.nih.gov/BLAST/.
Basic local alignment search tool (BLAST) is a sequence similarity search program
that can be used via a web interface or as a stand-alone tool to compare a users query to a
database of sequences. BLAST is a heuristic that finds short matches between two sequences
and attempts to start alignments from these hot spots. In addition to performing alignments,
BLAST provides statistical information about an alignment; this is the expect value, or
false-positive rate. The National Center for Biotechnology Information (NCBI) maintains a
BLAST server with a homepage at http://www.ncbi.nlm.nih.gov/BLAST/. On the homepage
the different BLAST searches are listed by type: nucleotide, protein, translated and genomes.
1. Comment on the conserved domain present in Q8NFM4.
Aim: To determine the conserved domain present in Q8NFM4
Introduction:
Conserved domains (CD) in proteins play a crucial role in protein interactions, DNA binding,
enzyme activity, and other important cellular processes. With recently released gene number
predictions in the human genome being less than many previous predictions, interactions among
these domains may prove to be central to proteome complexity. Protein domains are often
conserved across many species, and as such, they offer an interesting dataset in how genomes
maintain them with relationship to other conserved domains, as well as to proteome size.
Method:
1. Retrieve the sequence from NCBI.
2. Paste the sequence in the Query box in blastp.
3. Run against a non-redundant database (nr).
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Result and inference:
Query ID
gi|25008336|sp|Q8NFM4.1|ADCY4_HUMAN
Description
adenylate cyclase type 4 [Homo sapiens]
Specific hit] cd07302, cyclase homology
domain ;
Catalytic domains of the mononucleotidyl cyclases (MNC's), also called cyclase
homology domains (CHDs), are part of the class III nucleotidyl cyclases. This class
includes eukaryotic and prokaryotic adenylate cyclases (AC's) and guanylate cyclases
(GC's). They seem to share a common catalytic mechanism in their requirement for two
magnesium ions to bind the polyphosphate moiety of the nucleotide.
Blast Results:
Max score = 2214
Total score = 2214
Query coverage = 100%
E value = 0.0
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2. Find the gene sequences of Mouse origin similar to U80226.1.

Aim: To find the gene sequences of Mouse origin similar to U80226.1.
Introduction:
Sequence Similarity Searching is a method of searching sequence databases by using
alignment to a query sequence. By statistically assessing how well database and query
sequences match one can infer homology and transfer information to the query sequence.
Method:
1. Retrieve the Sequence of U80226.1
2. Enter the sequence in FASTA format in blastn
3. Choose Mouse genome+transcript as the Database.
4. Run blastn
Results and inference:
Similar Sequence: NM_172961.3

Mus musculus 4-aminobutyrate aminotransferase (Abat), nuclear gene encoding mitochondrial
protein, transcript variant 1,
mRNA
Length=46
53
GENE ID: 268860 Abat | 4-aminobutyrate aminotransferase [Mus
musculus] Score = 1817 bits (2014),
Expect = 0.0
Identities = 1305/1501 (87%), Gaps = 2/1501
(0%) Strand=Plus/Plus
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3.Write the function of c7ae31. Find its orthologous proteins

Aim: To determine the function of C7AE31 and to find its orthologous proteins.
Introduction:
Orthologous proteins with the same function in different species, Orthologous proteins with
modified function in different species, Orthologous proteins with major modification of
function, Orthologous proteins that have lost their function, Orthologous proteins that have
gained additional functions, The three-dimensional structure of orthologous proteins,
Prediction of secondary structure of proteins, Prediction of the three-dimensional structure of
proteins, Detecting sequence homology of protein-coding genes.
Method:
1.
Retrieve the Sequence of C7AE31 from Uniprot.
2.
Enter the sequence in FASTA format in blastp
3.
Run the query against SWISS-PROT database.
C7AE31
O90371 POLS_ONNVI
Structural polyprotein (O'nyong-nyong virus (strain Igbo Ora))
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O90369 POLS_ONNVS
Structural polyprotein (O'nyong-nyong virus (strain SG650))
P22056 POLS_ONNVG
Structural polyprotein (O'nyong-nyong virus (strain Gulu))
4. Write the function of P80404. Find its paralogous proteins.
Aim: To determine the function of P80404 and its paralogous proteins
Introduction:
Paralogous means genes that have arisen from a common ancestor and are present in the
same genome. Paralgous may or may not have the same function. Paralogous proteins are
proteins that have arisen by gene duplication. The group of paralogous proteins that are
descended from a common ancestor by gene duplication is called a protein family.
Method:
1. Retrieve the sequence from NCBI.
2. Paste the sequence in the Query box in blastp.
3. Run against a non-redundant database (nr).
Query ID
gi|48429239|sp|P80404.3|GABT_HUMAN
Description
4-aminobutyrate aminotransferase, mitochondrial precursor [Homo sapiens]
Paralogous
protein:
AAB38510.1
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gamma-aminobutyric acid transaminase [Homo sapiens]

Score = 1000 bits (2585), Expect = 0.0, Method: Compositional matrix
adjust. Identities = 482/500 (96%), Positives = 483/500 (97%), Gaps =
0/500 (0%)
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Lab in Immunology,

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Lab in Immunology, rDNA Technology and Bioinformatics Manual

Never work alone in the laboratory.

Keep your work area clean & dry.

Tie back long hair.

Special precautions for working with heat or fire:

Lab in Immunology, rDNA Technology and Bioinformatics Manual

Use test tube holders to handle hot laboratory equipments.

Use only Pyrex glasswares for heating.

Make sure that Bunsen burner hoses fit tightly.

Special precautions for working with chemicals

Never taste or touch substances in the laboratory without specific instructions.

Never smell substances in the laboratory without specific instructions.

Use materials only from containers that are properly labeled.

Wash your hand after working with chemicals.

Mix heat generating chemicals slowly.

Special precautions for working with electrical equipment

Never touch electrical equipment with wet hands.

Never use broken or chipped glassware.

Lab in Immunology, rDNA Technology and Bioinformatics Manual

Special precautions for working with live or preserved specimens

a specimen while holding it in the air.

Safety cabinet should be used while working with microbes.

Lab coats should be worn during the work in the lab.

Disposable items should be collected and autoclaved.

with iodine alcohol mixture, and wrapped with sterile bandage.

Skin contamination requires washing with water and removal of contaminated

Lab in Immunology, rDNA Technology and Bioinformatics Manual

SEPERATION OF PLASMA AND SERUM FROM BLOOD

Lab in Immunology, rDNA Technology and Bioinformatics Manual

Blood Serum Preparation

Lab in Immunology, rDNA Technology and Bioinformatics Manual

ABO BLOOD GROUPING

Lab in Immunology, rDNA Technology and Bioinformatics Manual

Wash the slides and wipe with Ethanol.

Wipe your finger with Ethanol.

Prick it with needle(freash).

Spot blood on the slide in three positions.

Add anti-sera A,B&D on respective spot.

Mix properly and cheak for agglutination.

Lab in Immunology, rDNA Technology and Bioinformatics Manual

Lab in Immunology, rDNA Technology and Bioinformatics Manual

5. Add one drop each of O, H, AH and BH antigens to the remaining four

Lab in Immunology, rDNA Technology and Bioinformatics Manual

ASO LATEX TEST

ASO Antigen: A stabilized buffered suspension of polystyrene latex particles

ASO Negative Control: Human serum containing 0.1% sodium azide as

Sufficient disposable pipettes.

Glass test slide.

Lab in Immunology, rDNA Technology and Bioinformatics Manual

Lab in Immunology, rDNA Technology and Bioinformatics Manual

Bring all test reagents and samples to room temperature.

Discard the disposable pipette.

Re-wash glass slide for future use

Lab in Immunology, rDNA Technology and Bioinformatics Manual

Lab in Immunology, rDNA Technology and Bioinformatics Manual

Lab in Immunology, rDNA Technology and Bioinformatics Manual

Lab in Immunology, rDNA Technology and Bioinformatics Manual

Lab in Immunology, rDNA Technology and Bioinformatics Manual

Lab in Immunology, rDNA Technology and Bioinformatics Manual

Lab in Immunology, rDNA Technology and Bioinformatics Manual

COUNTER CURRENT IMMUNO ELECTROPHORESIS (CCIE)

Prepare 10ml of 1.5% agarose (.15g in 10 ml of buffer) in 1X electrophoresis