Practical Manuals

Of

BSc Biotechnology

Semester V & VI

rDNA

PRACTICAL MANUAL

SEMESTER V

EXPERIMENT NO-1

AIM:- culture and maintenance of bacteria (e.coli)

MATERIAL REQUIRED:- Chemical Required:-

1. Tryptone/ peptone

2. Yeast extract

3. Nacl

4. Naoh

5. Agar

6. Ethanol

GLASSWARE:-

1. Flasks (1 ltr, 10 ml)

2. Culture tubes

3. Petiplates

4. Bert glass rod

5. Beakers (250 ml)

EQUIPMENT:-

Autoclave

Orbital shaker

Laminar air flow

Incubator

Water bath set at 48-50ºC

Cotton plugs

Microtips

Turn table

Inoculating needles

Ph paper

Eppendiff tubes pipettes

Tissue paper

Gas lighter.

Match Box

Cabinet

Cotton plugs

PRINCIPLE:-

The study of any microbe requires that we can isolate it from a mixed natural

population and grow it in as pure culture in a suitable medium plating methods are

generally satisfactory for the isolation of bacteria. Once a pure culture is

developed, maintenance of such culture is required to ensure term viability and

genetic stability and to protect them from contamination during storage or transfers

M/o like yeast, bacteria and unicellular algae are routinely used for experimental or

industrial application. Many of them hare simple culturing requirements

facilitating their isolation and maintenance as pure culture. They can even be

grown in minimal medium with few supplements as compared to plants and animal

tissues/ culture. Our current understanding of fundamental biological process at

molecular level has resulted largely from study of microbes. An important attribute

of unicellular organism is that they can be spread as single cells on a solid medium

where they can be divide to from a clone of cells upon incubation. The resultant

colonies are visible to naked eye making visualization, selection and counting

simpler on the other hand single cells derived from multicellular organisms are

different to culture.

POUR PLATE METHOD:-

The overnight culture of E.coli and serially delude it.

Inoculate 50/100ml with a diluted culture

Pour 20ml of molten medium into pert plates

Replace the cover and the plate is gently rotated in a circular motion to achieve

uniform distribution of m/os

Repeat the step 2 and 4 for all dilutions

Learn the plates undistributed for 15-20 mins

Invert the pert plates and inoculate at 37ºC

Count all the colonies and calculate CFU per ml.

PROCEDURE:-

a) POUR PLATE METHOD:-

1. Take an overnight grown culture of E.coli and serially dilute it to yield

approx.. 30-300 colony forming units.

2. Inoculate 50/100 ml of labelled sterile petriplate with the diluted with

culture.

3. Pour 20 ml of molten medium cooled to 45ºC into the part plate.

4. Replace the cover and the plate is gently rotated in a circular motion to

achieve uniform distribution of m/o

5. Repeat the above steps for all dilutions to be plated.

6. Learn the plate undistributed for 15-20 min on a flat surface to allow the

medium to set.

7. Invert the pert plates and incubate the plates overnight at 37ºC.

8. Count all colonies and remember that the embedded colonies will be much

smaller than those that grow on the surface.

b) STREAK PLATE METHOD:-

1. Sterilize a platinum loop by heating it until red hot in a flame allow it to

cool. You can check for coolness by touching the agar at edge of plate.

2. Pick up a loop full of liquid in column or bacterial from the surface of an

agar plate and starting about one inch from edge of plate. Streak lightly back

and forth with the loop making close parallel steaks. A no of variations of

this technique are practical such as four way streak plate method.

It is important to realize that in this technique we gradually thin out the no.

of bacteria in each successive streak with the goal of obtaining isolated

colonies.

3. Replace the lid of Petridis.

4. Invert the plate and incubate the plates overnight at 37ºC.

5. Observe the plates for isolated the plates and the quality of streaking.

CULTING OF E.COLI IN LIQUID LB BROTH:-

1. Take a measured volume of LB medium in a culture tube (5ml) or in a flask

(20ml) in a 50ml flask.

2. Aseptically transfer 50ml of inoculum to culture tube (500/5ml medium).

3. Incubate the tubs overnight at 37ºC in an orbital shaker (set at 200 rpm).

4. Observe the liquid both for growth. The medium will be turbid if organism

has multiplied.

PRECAUTION:-

1. After pouring the medium you must incubate all plates and culture

overnight.

2. Incubate pert plates in an inverted position to prevent rapour.

3. Disinfect laminar air flow surface properly with alcohol soln.

4. Open sterile glassware and medium only inside the laminar flow chamber,

close to flame to prevent contamination.

EXPERIMENT-2

AIM:-

To extract genomic DNA from bacterial cells.

REQUIREMENTS:-

Centrifuge, incubator, veaker, conical flask, measuring cylinder, micropipette,

micropipette tips, thermometer, water bath, distilled water.

REAGENT REQUIREMENT:-

Solution A and B, protenase K, DNA, lysozyme, bacterial lyse buffer, wash buffer,

collection tubes for sample.

STEPS:-

DAY 1:-

Break pyophilised viol+ added 0.1ml LB broth

Streaking+inoculating in 5ml LB broth

Incubate the plates at 37ºC

DAY 2:-

Pick single colony from LB broth

Inoculate 5ml of sterile LB broth into 3 plates

Incubate for 36 hours

DAY 3:-

Gel bacterial pellet

Centrifuge 25ml of culture at 6000 rpm at 4ºC for 8 mints

Pipette out supernatant

Resuspend cells in 700 ml of solution A (RT)

Incubate at RT for 5 mints

PRINCIPAL:- the genetic material (genome) in bacteria is not very well organized

as compared to enkaryotic genome with which its highly condensed and is present

as nucleosomes. Hence extraction of bacterial genomic DNA is fairly simple.

3 major types of technique or combination of them are employed in isolation of

nucleic acids, differential solutibility, obsorption methods or density gradient

certrifugation. The choice of method depends on the source of DNA being isolated

and its applications is removal of protines. This is accomplished due to difference

in their chemical properties. Most nucleic acid isolated protocols involve the foll

steps:-

Cell lyses

Enzymatic treatment

Differential solubility

Precipitation

Cells lysis:- Nucleic acids (DNA & RNA) must be solubilized from cells or other

biological material. This solubilization is usually carried out under denaturation

using sodium dodecyl agents. These denaturing conditions efficiently solubilize.

The nucleic acids are generally do not adversely effect them. In addition the

denaturing conditions promote the removal of proteins during the subsequent steps

and inhibit activity of nuclear that degrade the nucleic acids.

Spin at 1000rpm for 10 munities

Collect 500 ml of supornatend in a fresh vial

Add 1 ml of absolute alcohol to 500 ml supernatant

Mix until strands of DNA pipette out

Centrifuge at 1000 rpm for 30 mints

Spoat the ppt DNA with tip and transfer in fresh vial

At 1000 rpm for 10 mints, add 0.5 ml 10% alcohol to DNA pellet

Add 100ml of solution B

Incubate for 10 mints at 55ºC-60ºC for 20-30 mints

Pipette out 2.5 ml of DNA sample supernatant into a fresh medium

Enzymatic treatment:- unwanted component (RNA and protein) are degraded by

enzymes generally include lysis buffer. Two types of enzymes are used:-

Proteins (Proteinase K):- to remove protein

RNAse (RNAase A):- to remove RNA

Differential Solubility:- Phenol extraction:- it is an organic solvent used to separate

proteins from DNA. It is mixed in equal volume with DNA. The two phases are

then separated by centrifugation and the upper aqueous phase that contains the

nucleic acid is retained portions are seen as floccuted material at interface.

Adsorption to solid gel in presence of high cone. Salts that remove water from

hydrated material in soln. polysaccharides and proteins do not adsorb to silica gel

and are thus removed. DNA is then aluted out under low salt cond free of RNA.

Precipitation:- DNA is precipitated from diluted soln with ethanol or isopropanol

in the presence of sodium potassium acetate (ph 5-5.5) when added to final from a

initial cone of 0.3m. sodium and acidic ph will neutralized the highly charged

phosphate backbone and promote hydrophobic interaction the ppt. DNA is then

collected by centrifugation. The DNA pellet is rinsed with 70% alcohol athanol to

remove anzymes axcess salts, dried and dissolved in an appx. Buffer.

PROCEDURE:-

Revival of bacteria strain:-

Day-1:-

1. Break open the lyophilized eial and resuspended the host by adding 0.7 ml

of sterile LB broth.

2. Streak a lapful of this suspension onto a LB plates and inoculate the

remaining.

3. Inoculate the plates at 37ºC for 16 hrs.

Preparation of bacterial cell plates:-

Day 2:-

1. Pick a single colony and inoculate into 5 ml of sterile LB broth.

2. Incubate in a shaker incubator set at 37ºC for 16 hrs.

Day 3:-

1. Centrifuge 1.5 ml of culture at 600 rmp at 4ºC for 8 min. to gel the bacterial

pellet.

2. Remove the culture supernatant carefully and discard it.

3. Resuspended the cells in 700 ml of soln. A at room temp.

4. Incubate at room temp for 5 mints and spin at 1000 rpm for 10 min.

5. Collect 500 ml of supernatant in a fresh vial acoid taking the pellet.

6. Add 1 ml of absolute alcohol to 500 ml of supernatant from step 5. Mix by

inverting the tube till white strands of DNA out are seen.

7. Centrifuge at 100 rpm for 30 min. and discard the supernatant.

EXPERIMENT NO-3

AIM:- to carry out the spectrophotometric analysis of genomic DNA.

REQUIREMENTS:- Isolated DNA sample, spectrophotometer.

PRINCIPLE:-spectrophotometric analysis of DNA sample is done because it can

detect the conc. Of DNA present in sample. The analysis also identified the

contaminations like phenol and protein present in sample. The amount of U.V. rays

absorbed by DNA sample is directly proportional to amount of DNA in sample.

Usually absorbance is taken at 260nm conc. Of DNA is determined by using the

fact that 260nm absorbance corresponds to:-

i) 50mg/ml of DNA.

ii) About 40mg/ml of single stranded DNA of RNA.

iii) About 20mg/ml at single stranded oligonucleotide absorbance is also taken

at 280 nm. Ratio of (A260/A280) provides an estimate of purity of DNA

sample.

iv) If the ration is 1.8-2:- pure DNA

v) If the ratio is greater than 2:- RNA

vi) If the ratio is less than 1.8:- Protein sample absorbance is taken at 24nm.

Which detect the presence of contaminants absorbance at 260nm doesn‟t

imply that sample has DNA organic also absorbs 260nm. If nucleic acid

are present then the absorbance of 260 nm will higher than 240nm and if

nucleic acids are not present than absorbance at 260 nm will almost be

equal to or that at 240nm.

Absorbance at 230nm tells us about the presence of aromatic aa-bcoz at 230

nm, arrogance is due to peptide of A260/A230 should almost be half of

A260/A230 be equal to 1.

The spectrophotometric reading of 4 DNA sample at different wavelengths is as

follows:-

DNA Sample O.D at r=260nm O.D at r=280nm Ratio 260/280

Plasrnid DNA 0.075 0.051 1.4

Bacterial DNA 0.057 0.051 1.11

Human blood I 0.063 0.035 1.8

Human blood II 0.051 0.060 0.85

RESULT:-

1. The value of plasmid DNA, bacterial DNA and human blood sample

indicates the presence of high level of impurities.

2. The value of human blood 1 (1.8) indicate a pure DNA sample which is

purest of all the sample tested.

PROTOCOL:-

1. 10ml of DNA sample was taken in an eppendoff tube containing 990ml of

D/W.

2. Absorbance was taken at 260nm, 280nm,230nm, 240nm, 270nm.

3. All the DNA samples were analyzed and reading noted.

4. A graph was plotted b/w absorbance and were length.

RESULT:-

1. All the samples should A260/A280 ratio, greater than 2, these small amount

of RNA is greater present.

2. Since, absorbance at 260 nm is higher than 240nm which implies that

absorption is purely due to nucleic acids and not because of organic solvent.

3. A260/A270 ratio is greater than 1 thus nucleic acid is not free of phenol.

A260/A230 is almost half of the A260/A280 ratio for pure DNA.

Absorbance at 230nm is due to peptide bonds. If A260/A230 ratio is greater

than A260/A280 ratio implies that proteins is less and vice versa. The

DNA/gm yield of tissue was found.

PRECAUTIONS:-

1. Spectrophotometer should be 1st calibrated with blank.

2. Curettes should be clean and wipe with tissue after use.

3. Pipetting should be done carefully.

EXPERIMENT NO-4

AIM:- Agarose gel electrophoresis

REQUIREMENTS:- Agarose gel, 1*TAE buffer, gel casting tray, combs, power

supply, horizontal electrophoresis appartatus, ethidium bromide, sample

(DNA/RNA, micropipette tips, dyes.

PRINCIPLE:- Agarose is a linear polysaccharide made up basic unit of galactose

and 3,6 anhydrogalactose. The gelling properties and 3,6 anhydrogalactose. The

gelling properties of agarose are attributed to both inter and intra hydrogen

bonding within and between long agarose chains. The cross linked structure gives

the gel good anti-conventional properties. The gelling temp of agarose gel is 28-

40ºC.

Electrophoresis is a technique used to separate changed molecules DNA is

negatively charged at neutral ph and when electric field is applied across the gel,

DNA fragments migrates size of DNA and agarose concentration, conformation by

DNA applied current. Bromphoenol blue and xyaline cyanole are used as the

colour markers to monitor the process of agarose gel electrophoresis bromphoenol

blue migrates at the same rate as DNA molecule of appromiately 800 base paris, in

2% agarose as 150 bp.xyaline cyanole in 1% agarose gel migrates about the same

rate as 4000 base pairs DNA fragements typically 0.005-0.03% of final conc. Of

xyaline cyanide is used.

Preparation of Agarose gel electrophoresis

Weight 0.5gm of agarose

Boil in 50ml of 1*TAE buffer

Boil till agarose dissolve completely to form a clear solution

Cool down a few degree at room temperature

Add 2ml of ethbr and mix gently

Gel solution was four gently through the sides of the casting tray

Tray left undisturbed for 45 mints

Comb removed and casting tray placed in a electrophoresis chamber

Submerged the gel in TAE (30ml) in electrophoresis chamber

PROCEDURE:- Preparation of 1% agarose gel:-

1. Prepare 1x TAE by diluting appropriate amount of 50ml of 1x TAE buffer.

2. Weigh 0.5g of agarose and add to 50ml of 1xTAE. This gives 1% agarose.

3. Boil till agarose dissolves completely and a clear solution is obtained.

4. Meanwhile place the comb of electrophoresis set such that it is

approximately 2cm away from the cathode.

5. Add EtBr to the molten agarose to a fial concentration of 0.5 mg/ml and mix

gently.

6. Pour the agarose solution in the contron part of the tan when the temp

reaches approx.. 60ºC. do not generate air bubble. The thickness of it should

be around 0.5 to 0.9cm. keep the gel undisturbed at room temp. for the

agarose to solidity.

ELECTROPHORASIS:-

7. Pour 1x TAE buffer into the gel tank till the buffer level stands at 0.5-0.8 cm

above the gel surface.

8. Gently lift the comb, ensuring the wells remain intact.

9. Connect the power cord to the electrophoresis power supply according to

convention rod, anode, black: cathode.

10. Load the 10ml of three different DNA samples in wells in the desired order

and record the order.

11. Set the voltage to 50v and switch on the power supply.

12. Switch off the power when the tracking bye from the approximately 1-2

hours.

13. After electrophonic, DNA samples can be visuals under or trans illuminator.

PRECAUTIONS:-

1. Ethidium bromide must be handled carefully as it is a carcirogen wear

gloves while handling etbr soln.

2. Thaw the DNA samples before loading on the agarose gel.

3. Light the comb gently otherwise the wells would get broken.

EXPERIMENT NO-5

AIM:- Restriction engzyme digestion of the isolated DNA with 6,5 and 4 cutters.

PRINCIPLE:-

Restriction enzyme activity:-

Bacteria are under constant attack by bacteriophages. To protect themselves many

types of bacteria hare developed defense mechanism in the form of enzyme called

endonuclease that chop up any foreign DNA. Since these enzymes restrict the

infection of bacteriophages. They are termed “Restriction endonucleases”. These

molecular scissors found in the bacterial cytoplasm can prove dangerous to the

cell, so bacteria protect their own DNA by mothylating the adenine or cytosine

bases. The methyl groups block the binding of restriction enzymes, but not the

normal reading and replication of genetic information DNA from an attacking

bacteriophage will not have these protective methyl groups and will be destroyed.

Together restriction enzyme and its modification, methltransferase from a

restriction modification (RM) system. Pour kinds of RM systems are known.

There are distinguished based on subunit composition. Kinds of sequence

recognition and cofactors needed for their activity. Most characterized enzyme

(about 93%) belong the type II class. They comprise the commercially available

restriction enzymes used for DNA analysis and other manipulating.

Place the vials containing restriction enzyme once

The vials containing substrate (+DNA) and 2x assay buffer

Prepare 3 different rxn mixture using ECORT digestion

Add 20ml of assay buffer to each of the three vials

The type II restriction enzymes recognize specific DNA sequence and cleared the

DNA at fixed locations at or near the recognizing site. They act as dimers, each

subunits recognizing the same 5‟ :- 3‟ nucleotide, sequence in complementary

DNA strand and hence are said to recognize pallindronic sequence. Before the

restriction site on a long DNA, it binds to a site amids+ a very large number of

non-cognate sites. The protein is then trans located from the initial site to the

recognition site, in presence of Mg2+, the enzyme undergoes a conformational

change, which kinds the helix and clears the DNA producing „blunt‟ or „sticky‟

ends.

However, many restriction enzymes out in an offset fashion to give. Sticky ends,

which have protruding 5‟ or 3‟ ends with unpaired bases depending upon the fool

sequence and clears each back bone b/w G and A bases goving 5‟ protruding ends.

5‟-GAATTC-3‟-------------5‟-G AATTC-3‟

3‟-CTTAAG-5‟ 3‟-CTTAA G-5‟

1. FOURCUTTER ENZYMES:-

Sau 3A1-Staphylococcus aureus

5‟-GATC-3‟

3‟-CTAG-5‟

After incubation is complete add 10ml of gel

Make electrophoresis unit ready

Sample added to well are in foll order Well 1. Sau3a1 well 2. Not I well 3. Eoor I

Content in each vial were mixed properly

10ml of sample was loaded into well

Electrophoresis of samples done for 1 hrs

After electrophoresis, gel was studied under Ur-trans illuminator

5‟----------------GATC---------------3‟

3‟ CTA G 5‟

2. FIVE CUTTER ENZYMES:-

ECORI

5‟--------GAATTC-------3‟-------5‟----G AATTC-3‟

3‟--------CTTAAG-------5‟ 3‟-CTTAA G-5‟

3. SIX CUTTER ENZYMES:-

NOT1 NOCARDIA OTITIDIS

5‟ GCGGCCGC 3‟

3‟ CGCCGGCG 5‟

5‟ GCGGCCGC 3‟

3‟ CGCCGGCG 5‟

PROCEDURE:-

1. Place the vial containing restriction enzymes (ECORI, San3A1, Not1) on

ice.

2. Thaw the vial containing substrate (cambda DNA) and 2x assay buffer.

3. Prepare 3 different reaction mixtures in 1.5ml vials usin the following

components.

Reaction 1 (ECORT digestion)

1DNA 20ml

2xAssay buffer 25ml

ECORI 3ml

Reaction 2 (Sau3A 1 digestion)

1DNA 20ml

2x Assay buffer 25ml

2 Sau 31

Reaction 3 (Not 1 digestion)

1DNA 20ml

2x Assay buffer 25ml

Not 1 3ml

4. Incubate all the vials at 37ºC for 1 hour.

5. After an hour add 5ml of gel of gel loading buffer to each of the vials to stop

the digestion.

6. Meanwhile, preparing 1% agarose gel for electrophoresis.

7. Load the digested samples, 10ml of control DNA and 10ml of r/M/V.

digestic in different wells. Note down the order of loading.

8. Electrophoresis the samples at 50-100r for 1-2 hours.

9. After electrophoresis DNA samples can be visualized under UV trans

illuminator.

EXPERIMENT NO-6

AIM:-

Agarose gel electrophoresis of digested fragment.

MATERIAL REQUIRED:-

Equipment- Microwave oven, U.V. Trans illuminator.

Glassware- Conical flask

Reagent- D.W, eth.Br.

Other Requirements- Micropipette, tips, 1.5 ml vials

PRINCIPLE:-

Agarose gel electrophoresis is a procedure used to separate DNA fragments based

on their molecular weight and it is an intrinsic part of almost all routine

experiments conducted in molecular biology.

PREPARATION OF AGAROSE:- Agarose is a linear polymer extracted from

seaweeds. Its basic structure is purified.

Agarose is a powder insoluble in water or buffer at room temp. but dissolves on

boiling molten soln of agarose is then paused into a mould and allowed to solidify.

As it cools, polymers cross-link with polymerization i.e. sugar polymers cross-link

with each other and cause the soln to gel, the density or pose size of which is

determiner by the conc. Of agarose.

Electrophorasis of DNA fragments:- Electrophorasis is a technique used to seprate

charged molecules DNA is negatively charged at neutral ph and when electric field

is applied across the gel, the DNA migrates towards the anode. Migration of DNA

through the gel is dependent upon:-

1. Molecular size of DNA

2. Agarose concentration

3. Conformation of DNA

4. Applied current

Matrix of agarose gel cuts as a molecular sierra through which DNA fragments

more on application of electric current. Higher conc. Of agarose gives firmer gels

i.e. spores b/w cross linked molecules is less and hence smaller DNA fragments

easily travel through these spores. As the length of the DNA inc. it becomes harder

for the DNA to pass through the spores b/w the cross linked molecular is more the

progress of gel electrophoresis is monitored by observing the migration of a visible

dye through the gel. Two commonly used dyes are xylene canola and bromophenol

blue that migrate at the same speed as double std. DNA of size 5000 bp and 300

bp. These dyes are re charged, low mol, weight compounds along with samples at

the start of electrophoresis. When dye reaches towards the anode, electrophoresis

terminated.

PROCEDURE:-

Preparation of 1% Agarose gel.

Day 1:- REVIVAL OF HOST:-

a) Prepare 1xTAE by diluting appropriate amount of 60x TAE buffer.

b) Weigh 0.5g of agarose and add to 50ml of 1xTAE. This gives 1% agarose.

c) Boil till agarose dissolved completely and a clear soln. is obtained.

d) Meanwhile place the comb of electrophoresis set such that it is approx.. 2an

away from the cathode.

e) Add etbr to the molten agarose to a final conc. Of 0.5 mg/ml and mix

gently.

f) Pour the agarose soln. in the central part of the tank when the temp. reaches

approx.. 60ºC do not generate air bubbles. The thickness of the gel

undisturbed t the room temp. for the agarose to solidity.

g) Pour 1x TAE buffer into the gel tank till the buffer level stands at 0.5 to

0.8m above the gel surface.

h) Gently lift the comb. Ensuring the wells remain intact.

i) Connect the power cord to the electrophoresis power supply according to

convention.

Red:- anode Black:- Cathode

j) Load 10ml of three different DNA samples in wells in the desired order and

record the order.

k) Set the voltage to 5v and switch on the power supply.

l) Switch off the power when the tracking dye from the wells reaches 3/4th of

the gel this takes approx.. 1-2 hrs.

m) After electrophoresis, DNA samples can be visualized under the trans

illuminator.

PRECAUTIONS:-

1. Ethidium brornide must be handled carefully as it is a carcinogen, wear

gloves while handling Etbr soln. and gets stained with Etbr.

2. Threw the DNA samples before loading on the agarose gel.

3. Lift the comb. Gently otherwise wells should be denatured.

ANIMAL TISSUE CULTURE

Practical Manual

Semester- V

INDEX

S.NO. EXPERIMENTS

1. Sterilization Techniques: Theory and Practical

a) Glassware sterilization

b) Media sterilization

c) Laboratory sterilization

2. Sources of contamination and decontamination measures.

3. Preparation of Hanks Balanced salt solution.

4. Preparation of Minimal Essential Growth medium.

5. Isolation of lymphocytes for culturing.

6. Isolation of rat macrophages from peritoneum for culturing.

EXPERIMENT NO-1

AIM:- Preparation and sterilization of glassware.

MATERIAL REQUIRED:-

1. Disinfectant-hypochlorite, 300 ppm available chlorine.

2. Detergent-7x, Decon

3. Soaking baths

4. Bottle brushes

5. Stainless steel baskets

6. Aluminum foil

7. Sterility indicators

8. Sterilizing oven (upto 160°C)

PROTOCOL:-

1. Collection and washing of glassware

i. Immediately after use, collect glassware into detergent containing disinfectant. It

is very important that glassware does not dry before soaking.

ii. Soak overnight in detergent.

iii. Rinsing:- a. Brushing, b. Machine

Rinse 3-4 time using deionized water and then sensing is done without detergent

with deionized or RO water.

iv. After rinsing thoroughly invert bottles etc. in stainless steel wire basket and dry

up side down. After that cap bottles with aluminum foil and cool.

2. Sterilization of Glassware:-

i. Attach small square of sterile indicating tape or other indicators labelled to

glassware and tape.

ii. Place glassware in oven with fan circulated air and temperature set to 160°C.

iii. Ensure that the packing is not very tight. Leave room for circulation of hot air.

iv. Close the oven check the temperature to 160° and seal the oven and leave it for

one hour. Time should be recorded.

v. After one hour, switch off the oven and allow it to cool with the door closed.

vi. Use glassware within 24-48 hours.

EXPERIMENT NO-2

AIM:- To perform media sterilization

THEORY:- most of the commonly used media are available for commercially but for special and

sterilize the media. Some may be auto cleaned while labile solutions like media, trypsin and

serum must be filtered through 0.2 porosity membrane filter.

There may be some microbial contamination in the media. The characteristic features of

microbial contamination are:-

1. Sudden change in PH, usually decrease with bacterial growth and slightly increase with

fungal growth.

2. Sometimes cloudiness appeal in the medium with a slight flame as scum surface or spot

on the growth surface that dissipates when flask is moved.

3. Under a low power microscope (around 10X) space between cells will appear granular

and may shimmer with bacterial contamination.

4. Yeast appear as separate round or void practical that may or may not be in budding rate.

5. Fungus produce thin filamentous mycelium and sometimes dense lamps of spores are

formed.

PROCEDURE:-

i) For heat stable solutions:-

For heat stable solution (i.e. the substance those constituents does not breakdown into

simples substances) components are sterilized by autoclaving them at 121°C for 15

mins.

ii) For heat liable solutions:-

For heat liable solutions, filtrations is the only method. Heat liable solutions are those

containing disaccharide or polysaccharide which on their autoclaving changes into

monosaccharaides. Thus, the method used for their sterilization is filtration.

Two types of filters used are:-

1. Absolute

2. Depth

PRECAUTIONS:-

1. Do not autoclave heat liable solutions.

2. Heat stable solution should be autoclaved for 15-20 mints for proper sterilization.

3. Microbial infection should be checked carefully, it is sometimes confused with

precipitates of co media that are mainly proteins.

EXPERIMENT NO-3

AIM:- To perform laboratory sterilization.

REQUIREMENT:- Chemicals used for disinfection are:-

Acids & esters:- Benzoic acid, sulphur dioxide, sulphoides and metabisulphides.

Alcohols:- Ethanol, isopropanol, benzyl alcohol, chloroform, chloroputanol, phenyl

alcohol, phenyl alcohol.

Fumigation:- Formaldehyde [H(CHO)], hydrogen cyanide (HCN), U.V. rays.

THEORY:- Sterilization of laboratory is very important as contamination may come from

laboratory environment. For this purpose, sterilization of the laboratory is done by different

methods.

1. Cleaning with disinfectants:-

A wide range of disinfectants are present in the market. These have the potential to kill

the different types of microbial forms. Thus autoclaving is not possible to avoid

contamination of cultures. Culture rooms are cleaned with the help of disinfectant.

2. Eradication with U.V. lights:-

U.V. light is also supported to kill all microbial forms. These culture rooms are lightened

with U.V. light before & after doing the practical work.

PRECAUTIONS:-

i. Remove the shoes after entering the incubation room.

ii. Sterilization of apparatus should be done carefully before the inoculation.

iii. Personal cleanliness is very essential in the laboratory. Avoid contamination by mouth

near the laminar air flow.

iv. Hands should be properly sterilized by 70% alcohol or ethanol.

EXPERIMENT NO-4

AIM:- Isolation of lymphocytes for culturing.

REQUIREMENTS:- Sterile phosphate buffer saline ficoll, histopaque of density 7.1 mg/l,

centrifuge tube, sterile pipette tips.

THEORY:- The most commonly used method for the separation of lymphocytes is the

sedimentation through high density medium of a monocular cells can be obtained by centrifuging

whole blood or the ficoll plate and the commercial preparation are from ficoll.

PROCEDURE:-

1. Collect peripheral blood in the vial containing anti-coagulant.

2. Dilute blood 1:2 with phosphate buffer saline (PBS) layer not more than 3 times. The

volume of the distilled water into the layer of ficoll.

3. Always hold a tube at an angle while carrying out the layering so that the ficoll and blood

do not get mixed.

4. Centrifuge at room temperature at 400 rpm for 30 mins.

5. Take care that the centrifuge doesn‟t accelerate so rapidly.

Mononuclear lymphocytes and monocytes are recovered at ficoll plasma interphase when

they from a white band. The erythrocytes sediment through ficoll plasma and from a pellet.

PRECAUTION:-

i. Layering should be done carefully.

ii. Carefully dilute the blood.

iii. Take care that centrifuge should not decelerate.

iv. Observe the white layer carefully.

EXPERIMENT NO-5

AIM:- To study the sources of contamination and decontamination measures in ATC lab.

REQUIREMENTS:-

Tissue culture media in addition to provide cells is also ideal substrate for the growth of micro-

organisms. So, it is necessary to sterile the media, culture vessels, tools and instruments and

surface disinfect the explant as well.

It is important to avoid cross contamination, (when contamination or bacteria is carried

from one object/flask/ petriplete to another).

The characteristic features of microbial contamination are:-

i. A certain change in pH usually occur with bacterial growth. There is slight change in pH

during fungal growth. There is either no or very little narration in pH during the

growth of yeast.

ii. Cloudiness in the medium is sometimes with the slight film or scum on the surface or the

spots on the growth surface that dissipates when flask is moved.

iii. Under low power microscope, spaces between cells will appear granular and may

shimmer with bacterial contamination yeast appears as separate, round or avoid

particles that may or may not be in the budding state. Fungus produces thin

filamentous mycelia and sometimes denser clumps of spores.

iv. Under high power microscope it may be possible to solve individual bacteria depending

upon its shape (round and coeci).

With the sli preparation the morphology of bacteria can be resolved at 1000 x.

Microbial infection sometimes may be consumed with the precipitates of media that

are mainly:-

Equipment & media Sources of contamination Decontamination measures

i. Manipulation pipetting Non-sterilized surface Clean working area of lab and also

and dispersing.

ii. Solutions:-

Non sterile reagents

and media

Dirty storage

conditions

Inadequate

sterilization.

iii. Glassware and

screw caps.

iv. Tools, instruments and

pipettes.

v. Culture/flask and

and equipment.

Spillage of necks and

outside of bottles and on

the working surface.

Pouching or holding

pipette too low.

Poor commercial supply.

Dust and spores from

storage

Ineffective sterilization

Contact with non-sterile

surface.

Invasion insects or dust.

Dust and aerosol

items present.

Swab properly with 70% ethanol.

Dispensing or transferring should be

done by pipettes.

Filter or autoclave before use

Test the filter before use.

Dry heat autoclave, sterilization.

Don‟t store the unsealed materials for

more than 24 hrs.

Sterilization by dry heat before use.

Rasterize instruments (70% ethanol)

Use screw caps in preference to

stoppers.

Wipe flasks and bottles with 70%

ethanol.

media bottles.

vi. Facilities, rooms and

air.

vii. Work surface

viii. While operating,

hairs, hands, cloths,

breathe.

ix. Hoods

x. Tissue sampling

xi. Co2 incubators

Dust and spillage.

Dust room skin, hair,

and clothing dropped or

blown into culture.

Infected at the source

during dissection.

Growth of molds and

bacteria in humid

atmosphere also on the

wall.

Filtered or clean air is used.

Wipe the floor and work surface

regularly.

Swab the work surface with 70%

ethanol.

Wash hands thoroughly.

Less talking, wear a mask, tie hails.

Check the filters.

Wash hands thoroughly, wear a mask,

tie hails.

Check the filters.

Check the tissue.

Do not bring the tissue directly to lab

and disinfect the tissue surface

properly.

Clean out weekly with detergent and

70% ethanol.

Add bactericide or fungicide,

humidified water.

EXPERIMENT NO-6

AIM:- Isolation of rat macrophages from peritoneum for culturing.

RESUIREMENTS:- 10ml syringe, ice cold PBS & 2% BSA, centrifuge, rat, scissors, vials

leishman‟s stain, microscope etc.

Composition of PBS & BSA

PBS (Phosphate buffer saline)

Na2HPO4.2H2O-----1.56 gm (Basic)

NaH2PO4.2H2O----1.78gm (Acidic)

Set the pH at 7.4 (100 ml)

Add NaCl (for 100ml – 0.9gm)

100 ml = 0.9x100/105=0.945gm

10ml of PBS + 0.1gm of BSA

PROCEDURE:-

i. The rat was killed with chloroform. The skin was drenched in 70% alcohol to sterilize.

ii. Infinite small incision was made in abdominal cavity skin and skin was sterilized.

iii. 10ml of solution of PBS & BSA (Bovine) serum albuminwas taken in syringe & carefully

injected by lifting peritoneum cavity.

iv. The peritoneum was adjusted slowly to suspend the fluid uniformly after 5 mins

withdraw as much fluid from body and pour it into vials.

v. Now centrifuge the vial containing fluid for 10 minutes at 200 rpm.

vi. Discard the pellet and suspend it in leishman‟s stain for 1-2 minutes.

PRECAUTION:-

i. The experiment should be performed under aseptic conditions.

ii. Rat should be properly washed with 70% alcohol to avoid any infection.

iii. Ice cold solution has to be injected.

iv. The vessel of the peritoneum should not get damaged while fluid is being injected in or in

other words, blood contamination must not occur.

EXPERIMENT NO-7

AIM:- Preparation of hank‟s balanced salt solution.

Liquid

HBSS

1X

HBSS 10X

WITHOUT

Ca & Mg

HBSS 1X

WITHOUT

phenol Red

HBSS 1X

WITHOUT

Ca & Mg

HBSS 10X

WITHOUT

Ca & Mg

& phenol

Red

Sodium Chloride 8000 80,000 8000 80000 8000

Potassium Chloride 400 4000 400 400 40

Potassium phosphate,

monobasic KH2 PO4

60 600 60 60 60

Glucose 1000 10,000 1000 1000 1000

Phenol red Na salt 10 100 - 10 -

Sodium phosphate, dibasic

NaH2PO4, anhydrous

48 479 48 48 47.86

Magnesium sulfate

ahhydrous, MgSO4

98 - 98 - -

Calcium chloride anhydrous 140 - 140 - -

Sodium bicarbonate 350 - 350 350 350

AIM:- Preparation of Hank‟s Balanced Salt Solution.

REQUIREMENTS:- Sodium Chloride, Potassium Chloride, Potassium Phosphate, Monobasic

(KH2PO4), glucose, phenol red, Na salt, sodium phosphate, dibasic Na2HPO4 anhydrous,

magnesium sulphate , anhydrous MgSO4, calcium sulphate, anhydrous sodium bicarbonate.

THEORY:-

Hank‟s balanced salt solution (HBSS) is used for a variety of cell culture applications, such as

washing cells before dissociation, transporting cells or tissue samples, diluting cells for counting

and preparing reagents.

The essential function of a balanced salt solution is to maintain PH and osmotic balance

as well as provide cells with water and essential inorganic ions. It is designed for use with cells

maintained in non-CO2 atmospheric conditions. For cell dissociation procedures, HBSS

modified is used, which is formulated without calcium and magnesium are generally used as

transport media, or for reagent preparation.

EXPERIMENT NO-8

AIM:- Preparation of Minimal essential growth medium.

COMPOSITION:-

INGREDIENTS mg/ml

INORGANIC SALTS

Calcium chloride dehydrate 265.00

Ferric nitrate monohydrate 0.100

Magnesium sulphate anhydrous 97.720

Potassium chloride 400.000

Sodium chloride 6400.00

AMINO ACIDS

Glycine 30.000

L-Arginine hydrochloride 84.000

L-Cystinedihydrochloride 62.570

L-Glutamine 584.000

L-Histidine hydrochloride monohydrate 42.000

L-Isoleucine 105.000

L-Lysine hydrochloride 146.000

L-Methionine 30.000

L-Phenyl alanine 66.000

L-Serine 42.000

L-Threonine 95.000

L-Tryptophan 16.000

L-Tyrosine disodium sat 103.790

L-Valine 94.000

VITAMINS

Choline chloride 4.000

D-CaPantothenate 4.000

Folic acid 4.000

Nicotinamide 4.000

Pyroxidal hydrochloride 4.000

Riboflavin 0.400

Thiamine hydrochloride 4.000

i-Inositol 7.20

OTHERS

D-Glucose 45.00.000

Phenol red sodium salt 15.900

EXPERIMENT NO-8

AIM:- Preparation of Minimal essential growth medium.

REQUIREMENTS:-

Inorganic salts, amino acids, vitamins and others like D-glucose, phenol red, sodium salt.

THEORY:- Dulbecco modified eagle medium (DMEM) is one of the most widely used

modification of eagles medium. DMEM is a modification of Basal medium eagle (BME) that

contains four fold concentration of amino acids and vitamins, additionally, the formulation also

includes glycine, serine and ferric vibrates. The original formulation contains 1000 mgs/l of

glucose and was originally used to culture embryonic mouse cells.

DMEM high glucose is a further modification of original DMEM and contains 450 mgs

glucose per liter. The various other cell lines including primary cultures of mouse & chicken

cells as well as various normal & transformed cell lines.

PROCEDURE:-

i. Suspend 13.3 gms in 900 ml tissue culture grade water with constant, gentle stirring until

the powder is completely dissolved. Do not het the water.

ii. Add 3.7 gms of sodium bicarbonate powder or 49.3 ml of 7.5% sodium bicarbonate

solution for 1 litre of medium and stir until dissolved.

iii. Adjust the pH to 0.2 to 0.3 pH units below the desired pH using IN HCl or IN NaOH

since the pH tends to rise during filtration.

iv. Make up the final volume to 1000 ml with tissue culture grade water.

v. Sterilize the medium immediately by filtering through a sterile membrane fitter with

porosity of 0.22 micron or less using positive pressure rather than vacuum to

minimize the loss of CO2.

vi. Ascetically add sterile components/ supplements as required and dispense the desired

amount of sterile medium into sterile containers.

vii. Store liquid medium at 2-8°C and in dark till use.

Practical Manuals

Of

Biochemical and Biophysical Techniques

Experiment no. 1

Aim: To demonstrate the principle of sedimentation using swing out rotor and angle rotor.

Principle: Centrifugation separation techniques are based upon the behaviour of particles in an

applied centrifugal field. If a solution of large particle is left to stand then the particles will tend

to sediment under the influence of gravity. Particles which differ in density or size or shape

sediments at different rates in a centrifugal field. The rate of sedimentation is dependent upon the

applied centrifugal field (G) being directed gradually outwards „X‟ which is determined by

angular velocity of rotor and radial distance of particle from axis of rotation.

G=w2r

Since one revolution of rotor is equal to 2II radius, the angular velocity of rotor is revolutions per

minute may be expressed as:

W=2II rev min-1/60

G=4II(rev min-1)2 r /3600

The centrifugal field is generally expressed as relative CF is multiple of RCF

RCF=4II2(rev. Min-1)2 r/3600*980

RCF=1.11*10-5(rev min-1)2 r

Sedimentation coefficient: the velocity of a particle pe unit centrifugal field is referred to as

sedimentation coefficient

S=V/w2r

V=sedimentation rate, w= angular velocity ,r= radial distance of the particle from the axis of

rotation.

Rotors: rotating unit of centrifuge is called rotor

Types of rotors: 1 fixed angle rotors 2 vertical tube rotors 3 swing bucket rotors

1. Fixed angle rotors: these rotors have holes within their body and one can slide the

centrifuge tube within these holes. Since the holes are at an angle between 14 to 40

degree to the vertical,the tubes and sample solution within also take the same angle.

Under the centrifugal field,the particles moves rapidly outwards. The particles then settle

down the wall and pellet is formed at the outermost point of the tube. Particles differing

only in their sedimentation characteristics can only be resolved in these

2. Vertical tube rotors: These rotors have holes within their body in which one can slide the

centrifuge tubes. These holes lie parallel to the rotor shaft and not an angle. The pellet in

such rotors will be deposited all along the outer wall of the tube.

3. Swinging bucket rotors: These rotors have buckets that swing out to a horizontal position

when the rotors accelerates. Even in the swinging bucket rotors, the particles strike the

walls of the tube and slide down. But these rotors are normally used for density grade.

EXPERIMENT-2

AIM: To identify lipids in a given sample by thin layer chromatography.

REQUIREMENTS: Glass plates(20×20 cm,3mm thickness),chromatographic glass jar with

vacuum,greased lid,capillary tube,silica gel-G,sample.

SOLVENT SYSTEM: Chloroform:methanol:water(65:25:4)

PRINCIPLE: The separation of compounds in thin layer chromatography(TLC) is based on the

differential adsorption as well as partitioning of analytes between the liquid stationary phase and

mobile solvent phase.

This technique is rapid as compared to paper chromatography.Molecules get separated between

the high unit stationary phase and non polar mobile phase. Hydrophilic analytes have no affinity

to polar towards mobile phase resulting in its factor,measurement and separation.The separated

analytes are identified y comparing their values to that of reference standard.

The Rf value of an analyte depend upon:

The solvent system

Degree of saturation of mobile phase in the chromatography chamber

Type of adsorbent

Temperature and humidity

Thus,the Rf value for an analyte is constant for a given set of experimental conditions.So,

Rf = Distance travelled by analyte / Distance travelled by mobile phase

Commonly used stationary matrix for TLC includes silica gel-G,silica gel-

H,alumina,cellulose.Normally glass, aluminium or polyester spots are used for coating the

stationary phase matrices.

PROCEDURE:

Place thoroughly cleaned and dried glass plate on a flat surface.

Weigh 5g of silica gel-G and add 100 ml of distilled water and thoroughly shook the

contents.

Immediately poured the slurry onto the glass plate.Silica gel-G should be uniformly

layered of thickness 25-75µm. Air dried the plates at room temperature.

Activated the coated TLC plates in hot air oven at 110oc for an hour.

Remove the plates from the oven and allowed to cool at a temperature in a dessicated

chamber. Spotted the activated TLC plates with the lipid sample, standard as well as

unknown with the help of capillary tube.

Develop the plates in the solvent system consist of chloroform,methanol and water.

The chromatography‟s chromatogram is developed by placing the TLC plate vertically in

TLC chamber saturated with mobile phase in such a manner that spotted edge dips in

the solvent system.

Run the chromatogram until the solvent front reaches the top edges of the plates.

Remove the plate and mark the solvent front as soon as the plate is removed from the

chamber.

Air dried the plates at room temperature.

Located the lipid spots by spraying iodine vapours.

Calculated the Rf value of lipid omponents in the sample and identified them by

comparing their Rf values with lipid standard.

PRECAUTIONS:

Thoroughly cleaned the glass plates. Do not touch or damage the coated area and

edge of the plates.

Thickness of silica gel should be uniform throughout the plate.

The slurry should be free of clumps.

OBSERVATIONS AND CALCULATONS:

Distance travelled by solvent = 14.2cm

Distance travelled by component A= 5.2cm

Distance travelled by component B= 5cm

Distance travelled by component C= 6cm

Distance travelled by component D= 6.5cm

Rf value = Distance travelled by analyte / Distance travelled by solvent

Rf values:

Component A = 5.2 / 14.2 =0.37cm

Component B = 5 / 14.2 = 0.35cm

Component C = 6 / 14.2 = 0.42cm

Component D = 6.5 / 14.2 = 0.45cm

EXPERIMENT-3

AIM: To separate amino acid mixture by paper chromatography.

REQUIREMENTS:

I. Whattman no.1 filter paper sheet.

II. Oven set at 105oc.

III. Developing solvent: Take butanol ,acetic acid and water in the ratio 4:1:5 in a separating

funnel and mix it thoroughly.Allow the phases to separate out completely.The upper

organic phase used as a mobile phase.

IV. Ninhydrin spray reagent: Prepare fresh by dissolving 0.2g of ninhydrin in 100ml acetone.

V. Standard amino acid: Prepare solution of authentic samples of amino acid such as

glycine,proline,aniline etc. (1mg/ml of 10% isopropanol).

VI. A sample containing mixture of unknown amino acid.

OBSERVATIONS:

Movement of solvent = 13.3cm

Movement of glycine = 3.7cm

Movement of proline = 6.1cm

Movement of tryptophan= 8.7cm

Rf value = Distance travelled by analyte / Distance travelled by solvent

Rf value of glycine = 3.7 / 13.3 = 0.27cm

Rf value of proline = 6.1 / 13.3 = 0.45cm

Rf value of tryptophan = 8.7 / 13.3 = 0.65cm

RESULT:

Rf value of glycine = 0.27cm

Rf value of proline = 0.45cm

Rf value of tryptophan = 0.65cm

PRINCIPLE:

The amino acid in a given mixture or sample are separated on the basis of the differences in their

solubilities and hence differential partitioning coefficient in a binary solvent system.

The amino acid with high solubilities in a stationary phase moves slowly as compared to those

with higher solubilities in the mobile phase. The separated amio acids are detected by spraying

air dried chromatogram with the ninhydrin reagent. All the amino acids give purple or bluish

purple colour on reaction with ninhydrin except proline and hydroxyl proline which give a

yellow coloured product.

PROCEDURE:

i. Take whattman no.1 filter paper sheet and laid it on rough filter paper throughout the

experiment , care should be taken not to handle he filter paper with naked hand.

ii. Folded the whattman no.1 filter paper about 2.25cm from the edge. Draw a line across the

filter paper with a lead pencil at a distance of 2cm from the second fold. Put circular

mark at this line at a distance of 2.5cm from each other.

iii. With the help of capillary tube or micro syringe apply about 20µl of solution on each

standard mark.

iv. Hang the filter paper in chromatography chamber. Care should be taken that the base line

should not get submerged when mobile phase is added otherwise the spotted material

would get dissolved in the solvent.

v. Removed the paper and marked the solvent front with lead pencil and let it dry at room

temperature.

vi. Sprayed the filter paper with ninhydrin reagent and after drying it at room temperature

transfer it to an oven at 105oc for 5-10 minutes.

vii. Blue or purple coloured spots would appear on the paper. Marked the boundary of each

spot with lead pencil.

viii. Measured the distance between the centre of the spots and also the distance of the

solvent front from the base line.

ix. Calculate the Rf value of standard amino acid as well as the given mixture.

PRECAUTIONS:

i. Do not touch the paper with naked handsbecause sweat on hands contain significant

amount of amino acids.

ii. The spots of applied sample should be as compact as possible. Larger the spots,poorer is

the resolution.

iii. At the time of fixing paper in chromatography chamber, it should be ensured that the base

line on which the sample is applied does not gets washed away in the solvent.

iv. Dry the paper thoroughly before spraying with the detection reagent. Wet paper may

interfere with the appearance of evenly shaped compact spots.

EXPERIMENT-4

AIM: Separation of proteins by affinity column chromatography.

REQUIREMENTS: ABTS[2,2-azino-bis-(3-ethylbenzthiozoline)-6-sulfonic acid] as a

chromogen,concavalin A-agarose,H2O2 as a substrate,elution buffer,sodium acetate

buffer,solution 1,solution 2,solution 3,test tubes, spectrophotometer, measuring cylinder,distilled

water,glass cuvettes,micropipettes,tips.

PRINCIPLE:

Affinity chromatography is a method of selectively and reversibly binding proteins to a solid

support matrix based on the fact that biological affinities exists between molecules e.g. antigen

with antibody.One of the component,the ligand is immobilized onto a solid matrix which is then

used to selectively purify the target protein including a complete ligand in mobile phase or

changing pH,then elute the target protein out.

The performance of affinity chromatography is determined by comparing the specific activity of

protein before and after purification. Specific activity of protein (enzyme) is defined as the

activity per mg of protein. In order to determine specific activity,enzyme activity and protein

concentrations are observed.

Enzyme activity of HRP will be determined using H2O2 as substrate and ABTS as a chromogen.

HRP acts on H2O2 to release nascent oxygen that oxidize ABTS to give coloured product. The

intensity of coloured product is measured by spectrophotometer at 725nm.

PROTEIN ESTIMATION BY LOWRY METHOD:

This method is based on both Bureat and folin cioncalteane reaction. Here the peptide bonds of

protein react with copper under alkaline conditions producing Cu+

which reacts with folin‟s

reagent to give blue colour due to the reduction of phosphomolybdenum. The intensity of colour

is dependent on the tyrosine and tryptophan content of the proteins.

PROCEDURE:

i. Resuspended an aliquote of crude sample in 1ml of sodium acetate buffer.

ii. Gently pipette 0.5ml of concavalin A-agarose suspension into the column and allow the

resin to settle.

iii. Equilibrate concavalin A-agarose with 5ml of sodim acetate buffer. Allowed the buffer to

drain out completely.

iv. Saved 0.1ml of the crude sample for determination of enzyme activity and protein

concentration.

v. Loaded the remaining 0.9ml of the crude sample on to the column,allowed it to drain off

completely.

vi. Washed the column with 1ml sodium acetate buffer. Added another 4ml of buffer to

wash out any unbound sample.

vii. Eluted the bound HRP by using 1ml of elution buffer. Collected the entire amount in a

test tube. Labelled this sample as elute.

ESTIMATION OF HRP ACTIVITY:

i. Prepared substrate solution by adding 2µl of H2O2 to 1 ml of ABTS solution.

ii. Using substrate solution,blank the reading to zero at 725nm.

iii. To 2990µl of substrate solution, added 10 µl of elute and mixed. Noted down the change

in absorbance at 725nm,exactly after 20 seconds of mixing the reagents.

iv. Diluted the crude sample 1:20(10µl of crude sample + 190µl of distilled water).

v. To 2990µl of substrate solution, added 10µl of 1:10diluted crude sample and mixed.

Noted down the change in absorbance at 725nm exactly after 20 seconds of mixing of

reagents.

vi. The activity of enzyme in both the crude and elute sample is calculated as follows:

Extinction coefficient of ABTS at concentration of 1µmole / µl = 19

Activity of enzyme in reaction mixture = „x‟* rv *df / Sv

= „y‟ µ/ml

Where (dA-725) = change in absorbance at 725nm after 20 seconds

U=ABTS units

rv = reaction volume (in ml)

Sv = sample volume taken for assay (in ml)

df = 1 for elute , 10 for crude sample

Estimated the total enzyme activity in crude and elute samples.

In crude : activity / ml * volume loaded onto the column

In elute : activity / ml * elute volume from the column

OBSERVATION AND CALCULATIONS:

Crude sample absorbance = 725nm

At t = 0

O.D. of crude sample = 0.039

At t = 20 sec

O.D. of crude sample = 0.074

Change = (dA-725) = t20 – t0

= 0.074 – 0.039

= 0.035

ELUTE SAMPLE

At t = 0

O.D. of elute = 0.050

At t = 20 sec

O.D. of elute = 0.085

Change = t20 – t0

= 0.085 – 0.050

= 0.035

ACTIVITY OF ENZYME IN FRACTION MIXTURE

Crude sample = 3*(dA-725) / 19

= 3*0.035 / 19 = 0.005µmol/min

Elute sample = 3*(t20-t0) / 19

= 3*0.035 / 19 = 0.005µmol/min

ACTIVITY OF ENZYME IN SAMPLE

Crude sample = X*rv*df / Sv

= 0.005*3*10 / 0.01

= 15µ/ml

Elute sample = X*rv*df / Sv

= 0.005*3*1 / 0.01

= 1.5µ/ml

TOTAL ACTIVITY

Crude sample = activity/ml * volume loaded onto the column

= 15 * 0.9

= 13.5

Elute sample = activity/ml * elute volume from the column

= 1.5 * 0.9

= 1.35

EXTINCTION OFYIELD AND RECOVERY OF HRP

Recovery = (Total activity in elute / Total activity in crude) * 100

= (1.35 / 13.5) * 100

= 10

Yield = Total activity in elute / Total volume

= 1.35 / 0.9

= 1.5

EXPERIMENT-5

AIM: Separation of protein by ion exchange chromatography.

REQUIREMENTS: Carboxymethyl cellulose,10X equilibrate buffer,10X wash buffer,5X elution

buffer,neutralizing solution,0.5M phosphate buffer,tube for mixing,centrifuge

column,spectrophotometer,chicken egg,distilled water, beakers, test tube ,column

stand,tips,micropipette,quartz cuvette.

OBSERVATION AND CALCULATIONS:

Elute sample at 280nm = 2.479

Crude sample at 280nm = 2.603

Crude sample at 260nm = 2.832

PROTEIN CONCENTRATION OF SAMPLES

Crude sample = (1.55 * A280) – (0.77 * A260 ) * Dilution factor

= (4.034) – (2.180) * 20

= 37.08 mg/ml

Elute sample = A280 * dilution factor / 2.55

= 2.479 * 10 / 2.55

= 9.72 mg/ml

YIELD

= Protein concentration of elute * Total volume of elute

= 9.72 * 1.5

= 14.58 mg

PRINCIPLE:

Ion exchange chromatography works on the basic principle that oppositely charged particles

attract each other. The stationary phase consist of fixed charges on a solid support. These charges

can be either positive or negative. Hence, there are two types of ion exchangers i.e. cation and

anion exchanger. Cation exchanger possess negatively charged ions and attract positivelycharged

molecules e.g. carboxymethylcellulose. Anion exchangers have positively charged groups that

attract negatively charged molecules and thud separates anionic molecules e.g.

Diethylaminoethyl cellulose.

Proteins are complex ampholytes i.e.they can have positive as well as negative charge.

Isoelectric point of a protein is the pH at which net electric charge on protein is zero.

In ion exchange chromatography,the solution containing protein of interest is applied to an ion

exchanger. Protein binding to an ion exchanger is dependent on net charge of protein at that

particular pH and on ionic strength of mobile phase. Bound particles are then eluted out from the

stationary phase by increasing the concentration of counter ion or by changing the pH which

alters the charge on protein. Weakly charged protein is displaced from stationary phase by

increasing the concentration of counter ion. This results in separation of proteins based upon its

net charge.

Extension of purification of protein can be determined by computing its specific activity. The

ratio of enzyme activity to the mass of proteins in the sample is usually expressed as unit of

activity per milligram of protein.

PROCEDURE:

Purification of lysozyme

i. Broken the egg with the help of spatula in the petriplate and collected egg white

separately.

ii. Poured 6ml of egg white in centrifugation tube and to it added equal volume of

distilled water. Mixed it gently to get a homogenous mixture such that avoiding

bubble formation.

iii. The pH of the homogenous mixture was adjusted to 7 by slow addition of

neutralizing solution.

iv. Centrifuged the egg white solution at 6000 rpm for 10 minutes and collected the

supernatant.

v. Saved about 0.5ml of supernatant for measurement of lysozyme activity. Labeled

this as crude sample.

Preparation of column

i. Washed the empty column with hot water.

ii. Fixed the column to the stand. Removed the top cap of the column and packed the

column with 25ml of carboxymethyl cellulose

iii. Removed the bottom cap and equilibrate the column with 50ml of 1X equilibrate

buffer.

iv. Loaded the egg white sample supernatant to equilibrate carboxymethyl cellulose

column.

v. Replaced the top and bottom caps of the column and incubated for 1 hour at room

temperature with intermittent mixing

vi. After an hour allowed the column material to settle. Slowly pipette out or

decanted the supernatant without disturbing the column.

vii. Washed the column with approximately 30-40 ml of 1X wash buffer.

viii. Eluted the lysozyme from the column using 15 ml of 1X elution buffer.

ix. Started collecting the elute in the test tube as 2ml fraction monitor O.D. at A280.

Pooled the fractions that shows A280 above. Labeled this tube as elute.

Estimation of protein concentration

i. Diluted the crude sample 20 times with phosphate buffer. Zero the

spectrophotometer against phosphate buffer blank.

ii. Measured the absorbance at A260 and A280.

iii. Protein concentration of crude sample is calculated as:

= (1.55 * A280) – (0.77 * A260) * Dilution factor

= x mg/ml

Elute

i. Diluted the elute 10 times with phosphate buffer.

ii. Zero the spectrophotometer against phosphate buffer blank.

iii. Measured the absorbance at A280.

iv. Protein concentration in elute can be calculated as:

= A280 * dilution factor / 2.55

= y mg/ml

Where dilution factor = 10 and 2.55 is extinction coefficient of lysozyme ,i.e. A280 of 1mg/ml

lysozyme is2.55.

PRECAUTIONS:

i. Clean the apparatus thoroughly before use.

ii. Prepare the solution carefully.

iii. Note down the O.D. very carefully before setting spectrophotometer zero.

Practical Manuals

Of

Bioprocess Engineering

EXPERIMENT NO-1

AIM:- To study growth curve of microorganisms.

MATERIAL REQUIRED:- Nutrient broth or LB medium, sterile petriplates, micropipettes,

cuvette, conical flask, sterile tips, culture- overnight culture of E-coli, spectrophotometer.

PRINCIPLE:- The increase in cell size and cell mass during the development of an organism is

termed as growth. The growth of organism is affected by both physical and nutritional factors.

The physical factors include pH, temperature, osmotic pressure, hydrostatic pressure and

moisture content of the medium in which the organism if growing. The nutritional factors

include the amount of carbon, nitrogen sulphur, phosphorus and other trace elements provided in

the growth medium. Bacteria are unicellular (single cell) organisms. When the bacteria reach a

certain size, they divide by binary fission, in which the one cell divides in a geometric fashion in

the actively growing phase. To study the bacterial growth population, the viable cells of the

bacterial should be inoculated on to the sterile broth and incubated under optimal growth

conditions. The bacterium starts utilizing the components of the media and it will increase in its

size and cellular mass. The dynamics of the bacterial growth can be studied by plotting the cell

growth (absorbance) versus the incubation time or log of cell number versus time. The curve thus

obtained is a sigmoid curve and is known as standard growth curve. The increase in the cell mars

of the organism is measured by using spectrophotometer. The spectrophotometer measures the

turbidity in the broth culture is directly related to the number of microoranisms present, either

viable or dead cells, and is a convenient and rapid method of measuring cell growth rate of an

organism, thus, the increasing turbidity of medium indicated increase of the microbial cell mars.

The amount of transmitted light through turbid broth decrease with subsequent increase in the

absorbance valve.

The growth curve has four district phases:-

i. Lag phase:-

When a micro-organism is introduced into fresh medium, it takes some time to adjust with the

new environment. This phase is termed as log phase in which cellular metabolism is accelerated,

cells are increasing in size, but the bacteria are not able to replicate and therefore no increase in

cell mass.

ii. Exponential or logarithmic (LOG) phase:- during this phase, the microorganism are in a

rapidly growing and dividing state. Their metabolic activity increase and the organism

begin the DNA replication by binary fission at a constant rate. The growth medium is

exploited at the maximal rate, the culture reaches the maximum growth rate and the

number of bacteria increases logarithmically (exponentially) and finally the single cell

divides into two, which replicate into four, eight, sixteen, thirty two and so on that is

20,2

1,2

2,2

3,-----2

4, n is number of generations).

OBSERVATION TABLE:-

S.No. Time O.D (at 600nm)

1. 0 min 0.010

2. 30 min 0.065

3. 1 hr 0.083

4. 1 he 30 min 0.282

5. 2 hrs 0.630

6. 2 hr 30 min 0.943

7. 3 hrs 1.074

8. 3 hrs 30 min 1.129

9. 4 hrs 1.908

10. 4 hrs 30 min 1.206

11. 5 hrs 1.226

12. 5 hrs 30 min 1.220

13. 6 hrs 1.205

iii. Stationary phase:-

As the bacterial population continues to grow, all the nutrients in the growth medium are used up

by the microorganism for their rapid multiplication. This results in the accumulation of waste

materials, toxic metabolites and inhibitory components such as antibiotics in the medium. This

sterile the conditions of the medium such as pH and temperature, thereby creating an unfavorable

environment for the bacterial growth. The reproduction rate will slow down, the cells undergoing

division is equal to the number of cell death, and finally bacterium stops its division completely.

The cell number is not increased and thus the growth rate is stabilized.

iv. Decline or Death Phase:-

The depletion of nutrients and the subsequent accumulation of metabolic waste products and

other toxic materials in the media will facilitate the bacterial to move on to the death phase.

PROCEDURE:-

i. An isolated colony of the organism was inoculated into 15 ml nutrient broth or LB

medium and kept for overnight incubation.

ii. Following day, the O.D. of this culture was measured at wavelength 600 nm in

spectrophotometer.

iii. The O.D. was checked at every 30 mints interval and recorded.

iv. Using this O.D. value, a satirized growth curve of the microorganism was plotted

(absorbance versus time).

PRECAUTION:-

i. Always wear lab coat & gloves when in lab.

ii. Spectrophotometer should be switched on 20 minutes before its usage.

iii. Cuvettes should be clean & dry.

iv. Record the absorbance carefully.

EXPERIMENT NO-2

AIM:- to determine the specific growth rate and generations time of a bacterium during

submerged fermentation.

REQUIREMENTS:-

Laura broth, flask, autoclave, laminar air flow, spectrophotometer, E.coli etc.

EXPERIMENT NO-2

AIM:- to determine the specific growth rate and generations time of a bacterium during

submerged fermentation.

REQUIREMENTS:-

Laura broth, flask, autoclave, laminar air flow, spectrophotometer, E.coli etc.

PRINCIPLE:-

i. Lag Phase

During this phase, all the cells are adjusting to their new environment, cellular metabolism is

accelerated resulting in rapid biosynthesis in rapid biosynthesis of cellular macromolecules,

primarily enzymes, the cell grow in size. There is no cell divisions and no increment in

number of cells.

ii. Log Phase:-

On this phase there is rapid exponential increase in population which doubles regularly until

a maximum number of cells are reached. The length of log phase varies depending on the

organism and composition of medium, the average growth may be estimated to last 6 to 22

hours.

OBSERVATION TABLE:-

S.No. Time O.D (at 600nm)

1. 0 min 0.011

2. 30 min 0.063

3. 1 hr 0.082

4. 1 he 30 min 0.280

5. 2 hrs 0.630

6. 2 hr 30 min 0.940

7. 3 hrs 1.070

8. 3 hrs 30 min 1.125

9. 4 hrs 1.905

10. 4 hrs 30 min 1.206

11. 5 hrs 1.244

12. 5 hrs 30 min 1.218

13. 6 hrs 1.201

iii. Stationary Phase:-

During this phase the number of cells that undergoing division is equal to number of cells

that are dying. There is no further increase in cell number and the population is maintained at

its maximum level for a period of times.

iv. Decline Phase:-

Because of continuing depletion of nutrients and building of metabolic wastes, the micro-

organisms die at rapid rate and uniform rate. The decrease in population close which will

increase during the log phase.

PROCEDURE:-

Preparation of inoculum culture, prepare LB (250/1000) and autoclave it inoculate with

E.coli culture. Let it grow for 6-8 hours and use the culture as inoculum for studying growth

kinetics.

Inoculate LB growth medium with 1% inoculum. Take 1 ml of culture fluid. Take O.D. at

600 nm after every 30 minutes. Plot the graph between O.D. and time.

GENERAL CALCULATION:-

From graph, u for different growth phase can be calculated-

EXPERIMENT NO-3

AIM:- to study the effect of temperature on the growth of microbes.

MATERIAL REQUIRED:-

LB broth, flasks, laminar air flow, E.coli, spectrophotometer, different temperature setups,

cuvettes.

OBSERVATION TABLE:- At Temp 4°C

Time O.D

0 min 0.010

30 min 0.051

1 hr 0.073

1 he 30 min 0.101

2 hrs 0.041

2 hr 30 min 0.063

3 hrs 0.038

3 hrs 30 min 0.064

4 hrs 0.023

4 hrs 30 min 0.103

5 hrs 0.021

5 hrs 30 min 0.011

6 hrs 0.011

THEORY:-

The natural environment of E.coli cells is the lower intestine of warm blooded animals. Its

optimum growth temperature is 37°C. The doubling time or generation time for the most

E.coli strains in a rich medium at 37°C is between 20-40 minutes. E-coli cells cannot grow

well at temperature is higher than 42°C. They can tolerate lower temperatures with the lower

growth rate. Protein synthesis slows at temperature lower than 37°C. In most cases the

temperature set on the incubator is the E-coli growth temperature. The E.coli growth

temperature will have some delay when the medium is kept at 4°C or shifted from 37°C to a

lower temperature.

For Temperature 31°C:-

Time O.D

0 min 0.011

30 min 0.271

1 hr 0.490

1 he 30 min 0.887

2 hrs 0.983

2 hr 30 min 0.952

3 hrs 0.801

3 hrs 30 min 1.908

4 hrs 1.803

4 hrs 30 min 0.907

5 hrs 1.907

5 hrs 30 min 1.906

6 hrs 1.000

For Temperature 50°C:-

Time O.D

0 min 0.011

30 min 0.051

1 hr 0.043

1 he 30 min 0.101

2 hrs 0.109

2 hr 30 min 0.091

3 hrs 0.053

3 hrs 30 min 0.087

4 hrs 0.078

4 hrs 30 min 0.047

5 hrs 0.035

5 hrs 30 min 0.027

6 hrs 0.010

PROCEDURE:-

i. Prepare 1% LB broth by adding 4 G of powdered LB broth in 396 ml of distilled water.

ii. Autoclave the LB broth at 15 psi at 121°C for 15 minutes.

iii. Allow the media to cool and pour 50 ml of media in each flask.

iv. Inoculate the media with E.coli in the laminar air flow cabinet.

v. Provide different temperatures to the media, i.e., 4°C, 37°C and 50°C. keep one of the

flasks at room temperature.

vi. Note the absorbance of each sample after every 30 minutes at 600 nm.

PRECAUTIONS:-

i. Inoculation should be done under sterile conditions.

ii. Turn on the spectrophotometer 20 minutes before noting the absorbance.

iii. Perform inoculation near the flame of Bunsen to prevent contamination.

RESULT:- Maximum growth of E.coli is observed at 37°C. below and above this temperature,

the growth of E.coli is retarded.

EXPERIMENT NO-3

AIM:- to study the effect of pH on growth of microbes.

REQUIREMENTS:- LB broth, flasks, laminar air flow, E.coli, spectrophotometer, cuvettes,

glouicle acetic acid, NodH, pH meter, dropper, cotton plug and autoclave.

THEORY:- In addition to nutrients, the pH of the growth medium is also important for E-coli

growth rate and cell density. The optimum growth pH for E.coli is near neutral. E-coli of three

pH units (from pH 5.5 to 8.5). extreme pH beyond this range will significantly decrease the well

growth rate and may sometimes even cause cell death. The minimum and maximum growth pH

for E.coli are pH 4.4 & pH 9.0 respectively. E.coli cells appear to tolerate a low pH better than a

high pH. In fact, extended exposure of E.coli cells to a high pH cause cell lysis. At the saturation

or stationary phase, the pH of the E.coli culture in commonly used media is near its pH limits.

The medium pH is determined by medium compositions, buffers, cellular metabolites, and

aeration conditions.

E.coli cells use sugars such as glycerol and glucose as carbon or energy source. When E.coli

cells use these sugars as carbon source, they will produce acetic acid and therefore lower the pH

medium. Carefully balancing the phosphate buffer, organic buffers, sugar contents and aeration

conditions can maintain the culture medium near E.coli optimum growth pH or within range of

three pH units.

For pH 4:-

Time O.D

0 min 0.011

30 min 0.041

1 hr 0.131

1 he 30 min 0.123

2 hrs 0.094

2 hr 30 min 0.083

3 hrs 0.079

3 hrs 30 min 0.050

4 hrs 0.042

4 hrs 30 min 0.039

5 hrs 0.034

5 hrs 30 min 0.021

6 hrs 0.010

For pH 7:-

Time O.D

0 min 0.011

30 min 0.159

1 hr 0.231

1 he 30 min 0.457

2 hrs 0.663

2 hr 30 min 0.671

3 hrs 0.749

3 hrs 30 min 0.804

4 hrs 0.787

4 hrs 30 min 0.801

5 hrs 0.761

5 hrs 30 min 0.789

6 hrs 0.661

PROCEDURE:-

i. Prepare 1% LB broth by adding 4g of powdered LB broth in 396 ml distilled water.

ii. Autoclave the media at 15 psi pressure for 15 minutes at 121°C.

iii. Allow the media to cool and pour 50 ml of media in each flask.

iv. Inoculate the media with E.coli in the laminar air flow cabinet.

v. Media was provided with the different pH, i.e. , 4, 7 and 9.

vi. pH 4 was set by adding glacial acetic acid to the media and pH 9 by adding NaoH.

vii. Note the absorbance of each sample after every 30 minutes at 600 nm.

PRECAUTIONS:-

i. Inoculation should be done under sterile conditions.

ii. pH should be set accurately.

iii. Absorbance should be noted carefully and should be taken at 60nm.

For pH 9:-

Time O.D

0 min 0.011

30 min 0.290

1 hr 0.557

1 he 30 min 0.791

2 hrs 0.850

2 hr 30 min 0.971

3 hrs 1.305

3 hrs 30 min 1.809

4 hrs 1.101

4 hrs 30 min 1.789

5 hrs 1.503

5 hrs 30 min 1.204

6 hrs 0.832

RESULT:-

E.coli bacteria grow best at a ner neutral pH of 6-8. They do not grow at a more acidic pH of 4 or

5 and shows maximum growth at pH 9.

EXPERIMENT NO-3

AIM:- To study the effects of aeration on the growth of microbes.

REQUIREMENTS:-

LB broth, flasks, laminar air flow, E.coli, spectrophotometer, cuvettes, cotton plugs, parafilm,

cello-tape, autoclave.

THEORY:-

E-coli cells produce large quantities of acetic acid if the growth medium contains little or no

oxygen causing the growth medium to reach pH 4 or lower. At this pH, E.coli cell growth slows

down or even stops. Acetic acid is the major metabolic inhibitor under anaerobic growth

condition. However, with proper aeration, E.coli cells will be able to use many organic acids

such as carbon source and pH of the growth medium will be maintained at near neutral or basic

ranges. Aeration is another important factor condition allow the cells to use organic acids as

carbon source and increase medium pH. Selection aeration conditions can also help cell maintain

its medium pH.

For minimum Aeration:-

Time O.D

0 min 0.010

30 min 0.051

1 hr 0.101

1 he 30 min 0.087

2 hrs 0.048

2 hr 30 min 0.093

3 hrs 0.084

3 hrs 30 min 0.075

4 hrs 0.049

4 hrs 30 min 0.101

5 hrs 0.039

5 hrs 30 min 0.021

6 hrs 0.010

For optimum Aeration:-

Time O.D

0 min 0.011

30 min 0.109

1 hr 0.371

1 he 30 min 0.451

2 hrs 0.611

2 hr 30 min 0.733

3 hrs 0.691

3 hrs 30 min 0.805

4 hrs 0.783

4 hrs 30 min 0.791

5 hrs 0.774

5 hrs 30 min 0.749

6 hrs 0.634

PROCEDURE:-

i. Prepare 1% LB broth by adding 4g of powdered LB broth in 396 ml of distilled water.

ii. Autoclave the media at 15 psi pressure for 30 minutes at 121°C.

iii. Allow the media to cool and pour 50 ml of media in each flask.

iv. Inoculate the media with E-coli in laminar air flow cabinet.

v. Media in the flasks were given different aeration conditions.

vi. Minimum aeration is provided by firstly plugging the flask and then sealing it using

cello-tape, optimum aeration is provided by plugging the flask with cotton plug and

sealing with parafilm. Maximum aeration is provided by only plugging the flask with

cotton plug.

vii. Note the absorbance of each sample after every 30 minutes at 600 nm.

PRECAUTIONS:-

i. Inoculation should be done properly.

ii. Conditions should be provided careful.

iii. Cuvettes should be clean and dry.

iv. Wavelength should be set at 600 nm.

For optimum Aeration:-

Time O.D

0 min 0.011

30 min 0.383

1 hr 0.681

1 he 30 min 0.752

2 hrs 0.945

2 hr 30 min 0.961

3 hrs 0.949

3 hrs 30 min 0.904

4 hrs 1.805

4 hrs 30 min 1.701

5 hrs 1.203

5 hrs 30 min 1.604

6 hrs 0.903

RESULT:- Maximum growth is observed in the medium provided with maximum aeration,

growth is reduced at optimum aeration conditions and growth stops at the minimum aeration

conditions.

Practical Manuals

Of

rDNA-B (Semester-VI)

INDEX

S.No. EXPERIMENTS

1. Isolation of Plasmid DNA.

2. Digestion of plasmid with three different restriction enzymes.

3. Preparation of Competent Cells.

4. Transformation of Competent Cells by CaCl2 method.

5. Confirmation of the transformants for the presence of Plasmid.

6. Southern Blotting.

EXPERIMENT-1

AIM: Isolation of Plasmid DNA.

PRINCIPLE: Plasmids are double stranded circular self- replicating extra chromosomal DNA

molecules. They are commonly used as cloning vectors in molecular biology. Many methods are

used to isolate plasmid DNA essentially involving the following steps:

Growth

Harvest & Lysis of bacteria.

Isolation of plasmid DNA.

Alkaline lysis method for purification of Plasmid exploits the topological difference between

plasmid circles and linear chromosomal fragments. Cells are lysed by treating with alkali

(NaOH) & a detergent SDS. SDS denatures bacterial proteins and NaOH denatures the plasmid

and chromosomal DNA. However, in the case of plasmid the strands remains closely circularized

since they are linked by inter-ionized back bones of double helix DNA. In contrast, stands of

linear/nicked DNA are free to separate completely when this mixture of denatured plasmid and

chromosomal DNA is neutralized by the addition of sodium acetate. Renaturation of plasmid is

rapid and accurate since, the strands are already in close physical proximity. Linear molecules

generated by random shearing of chromosomal DNA renature less accurately forming networks

of DNA that can be removed from lysate by centrifugation together with denatured proteins and

RNA & can be precipitated using Ethanol/ Isopropanol. Agarose Gel Electrophoresis can be

performed later, to separate plasmid DNA by its size and shape.

REQUIREMENTS: Incubator, shaker, micro-centrifuge, vortex mixer, beaker, conical flasks,

test-tubes, measuring cylinder, petri-plates.

REAGENTS USED: Distilled water, crushed ice,

Solution I: maintains pH, preventing immediate lysis of cell.

50mM glucose

25mM Tris-Cl (pH- 8.0) &10mM EDTA (pH-8.0)

Solution II: SDS- an alkali & a detergent that disrupts cell membrane and denatures

chromosomal and plasmid DNA.

0.2N NaOH (freshly diluted from a 10N stock)

1% (w/v) SDS

Prepare solution II fresh and use at room temperature.

Solution III: a buffer which renatures the plasmid DNA.

5M potassium acetate -60 ml

Glacial acetic acid - 11.5 ml

H2O - 28.5ml

Solution IV: (Isopropanol) an alcohol which precipitates plasmid DNA.

RNAase A: degrades RNA without affecting DNA.

Luria broth, Ampicillin, Bacterial lyophilized culture (E.coli).

PROCEDURE:

Day 1: Revival of Host.

1) Break open the lyophilized vial and resuspend the sample by adding 0.1ml of LB broth.

2) Streak a loopfull of this suspension onto two LB plates containing ampicillin at a

concentration of 100µg/ml and inoculate the remaining suspension in 5ml LB broth with

ampicillin.

3) Incubate the plate at 370C overnight.

Day 2: Incubation of growth in shaker.

4) Pick a single colony for the LB plate and inoculate into 10ml LB broth containing

ampicillin (100µg/ml).

5) Incubate in a shaker set at 370C overnight.

Day 3: Plasmid Preparation.

Begin Alkaline Lysis as outlined below:

6) Pipetted 1.5ml culture each into 5 individual 1.5ml vials.

7) Spin at 6000 rpm for 8-10 minutes at 40C. Discard the supernatant and invert the vial on

blotting paper to drain out left over supernatant. Place on ice.

8) Resuspended cell pellet in 100µl of ice-cold solution I. vortex gently &place on ice for 5

minutes and shift to room temperature.

9) Added 200µl of solution II at room temperature. Mix gently by inverting the vial stores.

10) Added 150µl of solution III. Mix gently by inverting the vials. Place on ice for 5 minutes.

11) Spin at 6000-8000 rpm for 15minutes at 40C.

12) Transferred the supernatant immediately to a fresh vial and 450µl of solution IV to

precipitate the DNA. Mix by inverting the vial. Incubate at room temperature for 10-15

minutes.

13) Spin at 10,000 rpm for 20 minutes. Decant the supernatant. Inverted the vials on blotting

paper to drain out left over supernatant. DNA will be seen as white precipitate, sticking to

the side of the vial.

14) Dried the sample at 370C for 10-15 minutes till there is no trace of solution IV.

15) Resuspended the pellet in 20µl of 1X TE (added along the sides) mixed by tapping the

vial so that DNA goes into the solution.

16) Added 5µl RNAase.

17) Meanwhile, prepared 1% agarose gel.

18) Added 2µl of gel loading buffer to each of the 5 samples.

19) Loaded 20µl of extracted DNA (5 samples) along with 30µl of control DNA sample on

1% agarose gel & electrophoresis at100 volts for 1-2 hours.

20) Visualized the gel under U.V. transilluminator.

RESULTS:

Pink Colored Plasmid DNA bands as seen under U.V. transilluminator.

Electrophoresis Gel showing separation of dye.

EXPERIMENT-2

AIM: Digestion of plasmid with three different restriction enzymes.

REQUIREMENTS: DNA Sample, bromophenol blue dye, Ethidium bromide, restriction

enzymes (ECoRI, Hind III &BamHI), microcentrifuge tubes, pipettes, electrophoretic set up,

distilled water, gel.

PRINCIPLE: Bacteria are under constant attack of bacteriophages. To protect themselves many

types of bacteria have developed defense mechanisms in the form of enzymes called

endonucleases. These endonucleasescleaves any foreign DNA which the bacterial cell identifies

as foreign and cleaves it.because of this capacity it can be said that bacterium restricts the

progression of bacteriophages and thus these enzymes are called as Restriction Enzymes or

Restriction Endonucleases. These are used to obtain the restriction map of DNA, it helps to study

the arrangement of DNA. Also, it helps the geneticist to analyse population polymorphism & to

make desired modification in DNA strand to make recombinant DNA. These RE recognize

certain specific sequence known as recognition sequence within the DNA strand & cleave the

DNA at those specific recognition sequence. The RE produced two types of cuts: blunt cuts and

staggered cuts.

Some restriction enzymes with their recognition sequences are as follows:

Restriction enzyme Source (Organism & strain) Recognition sequence

ECoRI Escherichia coli Ry13 G/ AATTC

CTTAA/G

HindIII Haemophilus inflenzae Rd A/AGCTT

TTCGA/A

BamHI Bacillus amyloliquefaciens H G/CATCC

CCTAC/G

PROCEDURE:

1) Placed the vials containing restriction endonucleases (EcoRI, HindIII and BamHI) on ice.

2) Thaw the vials containing substrate (DNA) and 2X assay buffer.

3) Prepared three different reaction mixtures using the following constituents:-

a) Reaction 1 (EcoRI digestion)

Plasmid DNA -20µl

2X assay buffer - 25µl

EcoRI - 3µl

b) Reaction 2 (Hind III digestion)

Plasmid DNA - 20µl

2Xassay buffer - 25µl

HindIII - 3µl

c) Reaction 3 (BamHI digestion)

Plasmid DNA – 20µl

2X assay buffer - 25µl

BamHI - 3µl

4) Incubated the vials at 370C for 1 hour.

5) Meanwhile, prepared 1% agarose gel for electrophoresis.

Electrophoresis of the DNA sample:-

Made the electrophoresis unit ready.

The samples that were added to the wells were in the following order:

Well 1- plasmid DNA (Control)

Well 2- Plasmid DNA sample digested by EcoRI

Well 3- Plasmid DNA sample digested by Hind III

Well 4- Plasmid DNA sample digested by BamHI

The content in each vials were mixed properly by tapping the vials.

10µl of sample were loaded into the wells.

Electrophoresis of the sample was done for 1 hour.

After electrophoresis, the gel was studied under U.V. transilluminator.

EXPERIMENT-3

AIM: Preparation of Competent Cells.

PRINCIPLE: the ability to take up DNA efficiently by most bacteria is limited in nature.

However, bacterial cells can be artificially induced to take up DNA by treating them with CaCl2.

Culture of such cells that are capable of taking up of DNA is said to be Competent. The

condition required to produce competence vary from species to species. The phenomenon of

competence has not been understood very well. It appears to result from changes in the cell wall

of bacteria. In the course of developing competence, receptors of either kind are formed or

activated on the cell wall, which are responsible for initial binding of the DNA. The complex

thus formed is resistant to DNAase.

REQUIREMENTS: Equipments: Centrifuge, shaker incubator, spectrophotometer, glassware,

conical flask, petri-dishes, pipettes.

Reagent: distilled water

Others: capped centrifuge tubes, crushed ice, cuvette tips, pipettes, water bath.

PROCEDURE:

Day 1: Revival of Host.

1) Break open the lyophilized vial, added 0.1ml of LB media.

2) Streak in duplicate a loop-full of suspension onto LB plates & incubate at 370C

overnight.

Day 2:

3) Inoculated a single colony into 5 ml LB medium & incubated at 370C (in a shaker)

overnight.

Day 3:

4) Inoculated 1ml of overnight culture into 100ml LB medium in a 1 litre conical flask &

incubated at 370C in a shaker. Grown until the OD A600 reaches 0.23-0.26, this takes

about 2-3 hours.

5) Chilled the culture flask on ice for 10-20 minutes.

6) Transferred the culture aseptically into sterile centrifuge tubes and spin down at 6000

rpm for 8 minutes, preferably in a refrigerated centrifuge at 40C or spin at room

temperature.

7) Discard the supernatant & to the cell pellet added approx. 15ml of cold 0.1M CaCl2

solution aseptically. Suspended the cell pellet gently in the solution using a pre-chilled

pipette. Care must be taken not to remove the tubes from ice during resuspension.

8) Placed the tube on ice for 30 minutes.

9) Centrifuged at 6000 rpm for 8 minutes either at 40C or room temp.

RESULT:

EXPERIMENT-4

AIM:Transformation of Competent Cells by CaCl2 method.

PRINCIPLE: Bacterial transformation is a process in which bacteria manages to uptake or bring

in free/ external DNA from the environment/ surrounding medium. The purpose of this technique

is to introduce a foreign plasmid DNA into bacteria and to use these bacteria to amplify the

plasmid DNA.

The ability to take up DNA efficiently by most bacteria is limited in nature and this can be

artificially induced to take up DNA by treating with calcium chloride.

PROCEDURE:

1) Discard the supernatant collected from previous experiment & re-suspend gently in

0.6ml of cold 0.1M CaCl2 solution.

2) Aseptically transfer 100µl of competent cells into pre-chilled vials.

3) Competent cells are now ready & should be used immediately for the transformation

experiment as the efficiency of transformation drops on storage at temperature higher

than -700C.

OBSERVATIONS:

PRECAUTIONS:

1) Maintain aseptic conditions while handling experiment.

2) CaCl2 solution should be chilled enough.

EXPERIMENT-5

AIM: Confirmation of the transformants for the presence of Plasmid.

PRINCIPLE:

SCREENING OF TRANSFORMANTS:

Selection of cells containing foreign DNA is done based on the selection marker carried by this

DNA.

Example: pUC plasmid has ampicillin resistance factor that enables only transformed cells to

grow on LB-Amp plates. Non- transformants, which are ampicillin sensitive, donot produce

colonies on the selective medium. Transformants& non-transformants are therefore easily

distinguished.

SCREENING OF RECOMBINANTS:

Identification of recombinants among the transformed cells is generally done by insertional-

inactivation. With most cloning vectors, insertion of DNA fragments into the plasmid destroys

the integrity of one of the genes present on the molecule. As a result, the characteristic coding by

the inactivated gene is no longer displayed by host cells & this is called insertional-inactivation.

REQUIREMENTS: Centrifuge, shaker incubator, spectrophotometer, conical flask, petri-plates,

spreader, distilled water, crushed ice, cuvette, tips, micropipette, water-bath.

PROCEDURE:

1) Added 5µl of the plasmid DNA to aliquots of 100µl of competent cells. Gently tap &

incubated on ice for 20 minutes (for the DNA to bind to cells). The remaining one aliquot

will not be transformed.

2) Heat shock the cells by placing the vials in 420C water bath for 2 minutes, then return

vials to ice to chill for 5 minutes.

3) Added 1ml of LB broth aseptically to the vial and incubated at 370C in a shaker for an

hour. This is to allow bacteria to recover and express the antibiotic resistance.

4) Labeled 3 LB-Amp plates with X-gal &IPTG as a, b& c. Pipetted 100µl of LB broth on

to each plate. Added 25, 50, & 100µl of transformed cells to plates a, b &c respectively.

Mixed well & spread thoroughly using a pipette or spreader.

5) Plated 100µl of competent cells that has not been transformed to check for any cell

contamination. Labeled this as control plate/non-transformed plate.

6) Incubated the plates at 370C overnight.

CALCULATIONS:

Transformation efficiency = No. of colonies x 1000/ amount of DNA plated (in ng)

= 124 x 1000/ 2.5

= 4.96 x 104cells/µg

RESULT:

EXPERIMENT-6

AIM: to perform southern blotting of DNA.

REQUIREMENTS: Nylon membrane, vacuum transferences, apparatus shaker, U.V.

transilluminator, micropipette, agarose gel electrophoresis.

PRINCIPLE: The southern blotting was identified by prof. E.M. Southern in 1975. In this

technique, the DNA from an agarose gel is transferred to a nylon membrane and this membrane

is then used for hybridization of DNA with a probe.

Transference of DNA from gel to membrane involves:

a) DEPURINATION: Agarose gel containing DNA is treated with 0.2N HCl so that the

fragments are de-purinated & fragments larger than 8Kb are also transferred.

b) DENATURATION: The denaturation solution purely denatures the ds DNA to ss DNA

so that hybridization can take place with the probe.

c) NEUTRALISATION: It neutralizes the pH so that hybridization can take place.

PROCEDURE:

1) Membrane of appropriate size slightly larger than that of gel was cut & activated in

distilled water for 5-10 minutes.

2) The vacuum transfer apparatus was set & the nylon membrane was placed followed by

gel.

3) Depurination of DNA strand was done using 0.25N HCl.

4) After depurination, the gel was washed with distilled water & then treated with 0.5N

NaOH& 1.5N NaCl for 30 minutes.

5) Neutralization of gel was done after denaturation by treating it with neutralizing solution

for 30 minutes.

6) Now, the vacuum pump was maintained & simultaneously buffer was poured over the gel

with a pipette. The transfer was carried out in 3 hours. After that the gel was removed &

pores were marked on the membrane by HB-Pencil.

7) Completion of transfer was checked by viewing the nylon membrane with U.V.

Transilluminator. Membrane was washed in neutralization solution, air dried & then

baked at 800C for 2 hours to fix the DNA to nylon membrane.

OBSERVATIONS: The membrane was observed under UV Transilluminator in which

smear of DNA was shown which shows the transfer of DNA has occurred. When the gel

after transfer was viewed under UV light, no bands were seen, indicating that transfer was

complete.

PRECAUTIONS:

1) While depurinating the gel, care should be taken not to depurinate. It should be done only

for about 10 minutes. The lines on the blot should be properly marked.

2) Continuous pouring of buffer should be done.

3) The gel should not be allowed to dry during transfer.