Immunology RDNA Bioinformatics Manual

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Lab in Immunology,

rDNA Technology andBioinformatics Manual

H e a d o f t h e D e p a r t m e n t

D e p a r t m e n t o f B i o t e c h n o l o g y

P G P C o l l e g e o f A r t s &

S c i e n c e , N a m a k k a l

Dr.B.Prakash.,Ph.D



Lab in Immunology, rDNA Technology and Bioinformatics Manual

Head of the Department Department of Biotechnology PGP College of Arts & Science,

Namakkal

1

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.





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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 equipment’s.

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 glassware’s 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 Glassware’s and other laboratory equipment’s

1. Become familiar with the names and appearance of all the laboratory equipment’s

you will use.

2. Never use broken or chipped glassware.

3. Make sure that all glassware’s 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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7. Use care handling all sharp equipment’s, such as scalpels and dissecting needles.

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 anti-

coagulant 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 acentrifuge 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 anti-

coagulant. 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 ofantibody 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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Ex.No: Date :

WIDAL TEST

INTRODUCTION:

Enteric fever specific agglutinins (antibodies) are detected in patients after 15 days of

fever. BCG vaccinated patient’s 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 β-

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 -

20ºC.

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 intothe 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 50-60◦c and 4ml/plate was poured onto 5 grease

free glass plates placed on a horizontal surface. The gel was allowed to set for30minutes.

3.

Wells were punched by keeping the glass plate on the template.

4. The wells were filled with 10 μl each of the antiserum and the corresponding

antigens.

5. The glass plates were kept in a moist chamber overnight at 37◦c.

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. Don’t add excess

buffer.

7. Add 10µl of antigen in each of the 4 wells towards cathode (negative electrode)

and 10µl of antisera (1, 2, and 3) and 10µl 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 froththe 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 10µl 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 50μl 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 10μl antibody HRP conjugate.

Mix thoroughly .





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24

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:





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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 37°C 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 37°C 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 120μl of 0.1 M CaCl2 and 80μl 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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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-λDNA 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 withampicillin.

pUC18 vectors have a short segment of E. coli DNA, which contains the regulatory and

coding sequences of Lac Z gene that codes for β-galactosidase enzyme. Isopropyl

thiogalactoside (IPTG) is an inducer of Lac Z gene expression. β -galactosidase reacts with

the chromogenic substrate 5-bromo-4-chloro- β-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 β-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 β -

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

• 42°C water bath





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• 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 -80ºC 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 37ºC for 1 hr

• Meanwhile, 40μl of IPTG and 40μl 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.





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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 three-

dimensional 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 µl 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%Ficoll – 25%

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 µg/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 ethidium-

stained 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 (Tris-

acetate-EDTA) and TBE (Tris-borate-EDTA). DNA fragments will migrate atsomewhat 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





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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 DH5α 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 DH5α 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

minutes2. 875 µl of TE buffer is added to the cell pellet and the cells are resuspended in the

buffer by gentle mixing.

3. 100 µl of 10% SDS and 5 µl of Proteinase K are added to the cells.

4. The above mixture is mixed well and incubated at 37º C for an hour in an incubator.





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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 4º 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 µl 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 µl of TE buffer or distilled water is added.

14. 10 µl 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 :





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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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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 µl 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 µl 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 500µl 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 µl 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.





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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 µl 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 µl 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 μg of PUC18 DNA (10 ul) in a fresh eppendorf.

2. To this, add 11.5 µl of sterile water followed by 5 µl of 10X buffer.

3. Add 1.5 μl of BamH1 enzyme (1 units) and incubate the mixture at 37˚C for 2 hrs.





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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 :

(Incubate at 37˚ C for 1‐2 hrs)

RESULTS:

Materials Qty

PUC18 DNA 10 µl

Deionized water 11µl

10X buffer 3 µl

BamH1 1 µl

Total 25 µl





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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) Agarose4. 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.

1.5µl of PCR product of DNA fragment is added to reaction and – ve control vials

only.

3. 1 µl of 10X Ligation buffer is added to each of the three vials.

4. 1 μl of T4 D NA Ligase (1 U) is added to reaction and +ve control vials only.

6.5 μl, 8 μl, 7.5 μl of water is added to reaction, +ve control and – ve control vials,

respectively.

5. The total volume in each of the vials is 10 μl. Incubate for 1 hr at 37ο C.

6. The prepared mixtures can be analyzed in bacterial transformation in bacterial cells or

they can be analyzed onto agarose gel.

Incubate for 1 hr at 37ο C.

RESULTS:

Materials Qty

Double digested DNA (HIND III, BAM HI 2μl

10X Ligation buffer 1μl

T4 DNA Ligase 1μl

Water 6μl





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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 manyother 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





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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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Manual


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•

Genomics

•

Structural Bioinformatics

•

System Biology





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• Phylogeny/evolution

Services :





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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

NCBI’s 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 SNP’s:

This database contains data about SNP’s (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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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 the protein name

displayed on the 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

GRDKGKLSRLKPAPPPPPAASAGKAGGKPSQSPSQEAAGEAVLGAKTKATSLVDAVNSDAAKPSQPGEGLKKPVLPATPKPQSAKPSGTPISPAPVPSTLPSASSALAGDQPSSTAFIPL

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 user’s 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, orfalse-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 oftenconserved 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 IDgi|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 querysequences 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,

mRNALength=46

53

GENE ID: 268860 Abat | 4-aminobutyrate aminotransferase [Mus

musculus] Score = 1817 bits (2014),

Expect = 0.0Identities = 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.

Result and inference:

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).

Result and inference: Query ID

gi|48429239|sp|P80404.3|GABT_HUMAN Description

4-aminobutyrate aminotransferase, mitochondrial precursor [Homo sapiens]

Paralogous

protein:

AAB38510.1




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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Immunology RDNA Bioinformatics Manual