final report i

8/3/2019 Final Report i

1/61

A REPORT ON

ADVANCED BIOTECH TECHNIQUES

MASTER OF TECHNOLOGY

IN

BIOTECHNOLOGY

BY

RAGHVENDRA PRATAP SINGH

ONE MONTH TRAINING

FROM

21JUNE TO 20JULY-2011

UNDER THE SUPERVISION

Mr. JAHIR ALAM KHAN

RESEARCH SCIENTIST

MRD LIFESCIENCES

LUCKNOW


2/61

CHAPTER : I

MICROBILOGY


3/61


4/61

3. 6 test tubes were prepared, the first tube containing 5 ml of NaCl and the rest with 4.5 mlof it.

4. The Petri plates, test tubes and the nutrient agar medium were autoclaved for 15 minsunder a pressure of 15 psi pressure and temperature of 121 C.

5. After autoclaving pouring of nutrient agar media in Petri plates was done and the plateswere left until the media got solidified.

6. Then serial dilution was performed and 0.5 gm of soil sample was taken and mixed intofirst test tube then 0.5 ml of sample was pipette out from the first test tube and mixed into

the second test tube and the process continues till the last test tube.

7. From last tube 0.5ml of sample was taken and discarded.8. The soil sample was then spread on the media plates and the plates were e left for

incubation for 24 hrs in 37 c temperature.

Observation:

A number of colonies of the micro-organisms were visible on the Petri plates.

Result:

Petri plates with mixed cultures were obtained.


5/61

Precautions:

1. Handle the instrument carefully.2. Work in L.A.F. so as to avoid any contamination.3. The glass wares should be washed gently so that it might not infect the solutions stored in

them


6/61

Experiment no-2

Objective: To purify the obtained mixed culture.

Requirements: Petriplates containing the mixed colonies.

Principle:

In microbiology, streaking is a technique used to isolate a pure strain from a single species of

microorganism, often bacteria. Samples can then be taken from the resulting colonies and

a microbiological culture can be grown on a new plate so that the organism can be identified,

studied, or tested. The streaking is done using a sterile inoculation loop. This is dipped in

an inoculum such as a broth or patient specimen containing many species of bacteria. The sample

is spread across one quadrant of a petri dish containing a growth medium, usually an agar

plate which has been sterilized in an autoclave.

1.Streaking:

A. Quadrant: In quadrant streaking, streaking first strated from one end and simple lines are

made with the help of inoculation loop. Then plate is rotated 90 degree and again the procedure

is followed and then again plate is rotated 90 degree and streaking is done. After this in the

middle simple or zig zag streaking is done.

B. Discontinuous : In discontinuous streaking, first simple or zig zag streaking is performed

at one end and then the loop is heated for 3 to 4 seconds and cooled. Then the plate is rotated 90

degree and simple streaking is done. The loop is again heated for 3 to 4 seconds and cooled. The

plate is rotated 90 degree again and simple straking is performed.

2. Physical Characteristics: After streaking the colonies observed have different

morphology , like:

Colony Morphology:

SHAPE may be Circular, puntiform, filamentous, rhizoidal, irregular.
http://en.wikipedia.org/wiki/Microbiologyhttp://en.wikipedia.org/wiki/Bacteriahttp://en.wikipedia.org/wiki/Microbiological_culturehttp://en.wikipedia.org/wiki/Inoculation_loophttp://en.wikipedia.org/wiki/Inoculumhttp://en.wikipedia.org/wiki/Petri_dishhttp://en.wikipedia.org/wiki/Growth_mediumhttp://en.wikipedia.org/wiki/Agar_platehttp://en.wikipedia.org/wiki/Agar_platehttp://en.wikipedia.org/wiki/Autoclavehttp://en.wikipedia.org/wiki/Autoclavehttp://en.wikipedia.org/wiki/Agar_platehttp://en.wikipedia.org/wiki/Agar_platehttp://en.wikipedia.org/wiki/Growth_mediumhttp://en.wikipedia.org/wiki/Petri_dishhttp://en.wikipedia.org/wiki/Inoculumhttp://en.wikipedia.org/wiki/Inoculation_loophttp://en.wikipedia.org/wiki/Microbiological_culturehttp://en.wikipedia.org/wiki/Bacteriahttp://en.wikipedia.org/wiki/Microbiology


7/61

MARGIN may beentire, discrete, curled, lobate. ELEVATION may beflat, raised/ elevated, convex, convonate, umbonate. TEXTURE may be smooth, rough, hard, brittle, gummy. OPACITY : opaque, transparent. COLOUR may be yellowish, white , off white.

Procedure :

1. Purification of obtained mixed cultures was then done by the method of streaking.2. The method of streaking was followed by observing the colony morphology.

Observation :

Fig. : Quadrant streaking Fig. : Discontinuous streaking

Observation of colonies obtained in quadrant streaking :

A. Shape: REGULARB. Margin: ENTIREC. Elevation: ELEVATEDD. Texture: SMOOTH


8/61

E. Size : 0.1mmF. Colour : OFF-WHITE

Result: The obtained mixed culture was purified.

Precaution:

1. Handle the instruments carefully.2. Work in L.A.F so as to avoid any contamination.3. The glasswares should be washed properly so that it might not infect the solutions stored

in them.


9/61

Experiment no-3

Objective:To prepare a slant.

Requirements: Test tube, beaker, cotton plug, measuring cylinder, aluminium foil, pipette.

Nutrient agar (peptone- 5gm, beef extract- 5 gm, pH-7, agar- 20gm).

Principle : Slant culture is the one made on the surface of solidified medium in a tube which

has been tilted to provide a grater surface area for growth . Agar slant has many advantages.

Saves space. Suitable for long term storage. More media can be stored. Less contamination.

Procedure :

Nutrient agar medium (50 ml) was prepared. The media was poured in 6 test tubes , kept in a slant position and autoclaved. The process of streaking was done after the media got solidified. Streaking was done with help of inoculation loop, it should not touch the surface of the test

tube.

Observation: After 24hrs streaking result was observed as :


10/61


11/61

Experiment no- 4

Objective: To perform gram staining for identification of microorganisms.

Requirements: Slides, conical flask, aluminium foil, cotton plugs.

Apparatus used: Laminar air flow, microscope.

Reagents used: Crystal violet, iodine, water, safranine, ethanol.

Principle:

The crystal violet stain is the primary stain, which stains everything in the smear blue. The

Grams iodine acts as a mordant that causes the crystal violet to penetrate and adhere to the gram

positive organisms. The acetone-alcohol mixture acts as the decolorizer that washes the stain

away from everything in the smear except the gram-positive organisms. The safranine is the

counter-stain that stains everything in the smear that has been decolorized: pus cells, mucus,

gram-negative organisms. The gram-negative organisms will stain a much deeper pink.

Procedure :

A clean glass slide was taken and a thin smear of bacterial culture was made. Heat fixedand air dried.

Smear was flooded with crystal violet for 1 minute. It was washed with distilled water and air dried. The slide was flooded with grams iodine for 1 minute. It was washed with distilled water and air dried. 95% ethanol was added for 20 seconds. It was distilled with distilled water and air dried. Safranine was added for 30-45 seconds. It was washed with distill water and air dried. The slide was observed under microscope.


12/61

Observation:

Gram-negative bacteria, rod and cocci were observed.

Precaution:

1. Avoid air bubble on the slide.


13/61

Experiment no- 5

Objective: To determine sensitivity of bacteria to various antibiotics.

Principle:

The principle of antibiotic sensitivity test is based on the Bauer-Kirby disc diffusion method. It is

a standard qualitative test wherein the bacterial culture is spread onto the surface of Mueller-

Hinton agar, followed by addition of antibiotic impregnated discs to the agar surface. The

antibiotic diffuses through the agar to form a concentration gradient. This concentration gradient

influences the growth of the bacterial strain. If an organism is susceptible to an antibiotic, a clear

zone appears around the disc where the growth has been inhibited, called the zone of inhibition.

If resistant, no clear zone of inhibition appears. The diameter of the zone of inhibition

surrounding the antibiotic disc is measured to determine whether the micro-organism is sensitive

(S), intermediately sensitive (I) or resistant (R) to a particular

antibiotic. The size of zone of inhibition depends on:

The rate of diffusion of the antibiotic through agar and

The concentration of the antibiotic present in the disc.

Hence, by determining the susceptibility of a pathogen, clinicians can select the most appropriate

agent for treating the disease. The test also helps in studying microbial strains.

Requirements:

Equipment : Incubator (37C), Shaker.

Glassware : Conical flask, Measuring cylinder,

Petri plates, Test tubes.

Reagent : Distilled water.

Other Requirements: Forceps, Micropipette, Tips.

Procedure:

1. Prepare 15 Nutrient Broth (LB) agar plates.


14/61

2. Label the MH agar plates asE.coli, S. aureus and P. aeruginosa ..

3. Spread 50l of the three strains onto the respective labelled NB agar plates.

4. Make three wells on the NB agar plate.

5. Load antibiotics onto 2 wells and distilled water on 1 well.

6. Incubate at 37 degree centigrade for overnight in straight position.

7. Then observe result.

Observation:

Result:

Culture Antibiotic Concentrati

on

Volume Zone of

Inhibition(in

cm)

E.coli Ampicillin

Tetracyclin

Cefixine

1mg/ml

100g/ml

1mg/ml

100g/ml

1mg/ml

50l

50l

50l

50l

50l

3

0

1.8

1.5

1.2


15/61

100g/ml 50l 0

Staphylococcus.aureus Ampicillin

Tetracyclin

Cefixine

1mg/ml

100g/ml

1mg/ml

100g/ml

1mg/ml

100g/ml

50l

50l

50l

50l

50l

50l

2.8

2.7

2.3

2.5

0

0

Pseudomonas.aeruginosa Ampicillin

Tetracyclin

Cefixine

1mg/ml

100g/ml

1mg/ml

100g/ml

1mg/ml

100g/ml

50l

50l

50l

50l

50l

50l

2.70

1.70

1.9

1.79

0

1.20

Antibiotic having maximum zone of inhibition for each culture is:

a. E.coli: 1mg/ml ampicillinb. Staphylococcus: 1mg/ml ampicillinc. Pseudomonas: 1mg/ml ampicillin


16/61


17/61

Experiment no- 6

Objective: Determine the Minimum Inhibitory Concentration of Staphylococcus for

Ampicillin.

Requirements: Test Tube, Beaker, Flask, Aluminium Foil, Cotton Plug, Pipette.

Chemical Required: Nutrient Broth, Ampicillin, Staphylococcus, Ethanol.

Requirements: Laminar Air Flow, Incubator, Autoclave.

Principle:

Minimum Inhibitory Concentration is the concentration at which the sensitive microorganism is

unable to grow or multiply. MIC determines the potency of the antibiotic or antimicrobial compound

against the test organism, it means the amount needed to inhibit the growth of a test organism is

required in low concentration.

Procedure:

1. Nutrient Broth was prepared (20 ml).2.

6 test tubes are taken and autoclaved.

3. 3ml of NB was poured in each test tube.4. 20l Staphylococcus was added in 5 test tubes and 1 test tube is taken as Blank.5. All the test tubes were then incubated in Shaker at 37 degree centigrade for 24 hours.

Observation:

Test Tube No. Culture Antibiotic Concentration(mg/ml) OD(at 600nm)

1 E.coli Ampicillin 0.25 0.01







18/61

Result:

The Minimum Inhibitory Concentration is=.00024mg/ml at OD 0.28.

Precaution:

1. Always keep LAF clean with alcohol do all the work carefully without contamination.2. Always clean hands with alcohol before working on autoclaved apparatus.


19/61

Experiment no- 7

Objective: To obtain bacterial growth curve.

Requirements: Petri plates, conical flask, measuring cylinder, cotton plug, pipette,aluminium foil, tip box.

Reagents: Nutrient broth(yeast extract- 5g, tryptone- 10g, NaCl- 10g, pH 7.2-7.5 for 1 litre).

Apparatus used: Autoclave, shaker incubator, colorimeter, laminar air flow

Principle: The doubling of every biochemical unit of cell within the time duration of a single

division without a change in the rate of growth results in bacterial growth curve.

During lag phase, bacteria adapt themselves to growth conditions. It is the period wherethe individual bacteria are maturing and not yet able to divide. During the lag phase of

the bacterial growth cycle, synthesis of RNA, enzymes and other molecules occurs.


20/61

Exponential phase(sometimes called the log phase or logarithmic phase) is a periodcharacterized by cell doubling . The number of new bacteria appearing per unit time is

proportional to the present population. If the growth is not limited, doubling will continue

at a constant rate so both the number of cells and the rate of population increases doubles

with each consecutive time period. For this type of exponential growth, plotting the

natural logarithm of cell number against time produces a straight line. The slope of this

line is the specific growth rate of the organism, which is a measure of the number of

division per cell unit time. The actual rate of this growth (i.e. the slope of the line in the

figure) depends upon the growth conditions, which effect the frequency of cell division

events and the probability of both daughter cells surviving. Exponential growth cannot

continue indefinitely, however , because the medium is soon depleted of nutrients and

enrich with wastes.

During stationary phase the growth rate slows as a result of nutrient depletion andaccumulation of toxic products. This phase is reached as the bacteria begin to exhaust

the resources that are available to them. This phase is a constant value as the rate of

bacterial growth is equal to the rate of bacterial death.

At death phase, bacteria run out o nutrients and dieProcedure:

Prepare 50 ml of LB media. Then autoclave this LB media. After autoclaving, inoculate 50 l ofE.coli culture. Incubate at 37C in shaker. Take the optical density day by day at 600 nm.

Observation:


21/61

DAY CULTURE O.D.

1 E.coli 1

2 E.coli 0.98

3 E.coli 1.15

4 E.coli 1.20

5 E.coli 1.18

6 E.coli 1.12

Result:


22/61

Experiment no- 8

Objective: To perform primary screening of active microbes.

Requirements: Petri plate, beaker, conical flask, tip box, pipette, cotton plug, aluminium foil,measuring cylinder, test tubes.

Reagents used: NaCl, nutrient agar media.

Apparatus: L.A.F., incubator.

Principle:

Primary screening is identification and isolation of active microbes. Microbes which are having

antibiotic properties, they will inhibit the growth of other microbes. These are actinomycetes

bacteria found in rhizosphere soil. All antibiotics are isolated from actinomycetes.

Procedure:

1. 6 Test tubes were prepared, the first tube containing 5 ml of NaCl and the rest with 4.5 ml ofit.

2.

Nutrient agar media was prepared.3. Test tubes, petri plates and NA media war kept for autoclaving.4. Serial dilution was performed.5. Nutrient agar media was poured in the petri plates.6. A nutrient agar plate was inoculated with soil sample containing active microbes for the

production of bioactive compounds 9antibiotics.

7. Plate is incubated at 37 C for 4-5 days.8. Then result is observed.


23/61

Observation:

Result:

There is clear zone around the colonies can be observed easily. The small colony inside

the clear zone (zone of inhibition) can be isolated for secondary screening of organism.


24/61

Experiment no- 9

Objective: To Perform Secondary Screening.

Requirements:

1. Equipment: Incubator (37C), Shaker.2. Glassware : Conical flask, Measuring cylinder,3. Petri plates, Test tubes.4. Reagent: Distilled water.5. Other Requirements: Forceps, Micropipette, Tips.

Princple:

Secondary screening is screening of culture, that it is producing antibiotics or not.

Procedure:

1. Prepare Nutrient agar plates.2. Spread 50l of the pathogens on NA plates.3. Make three wells on the NA plate.4. Load known antibiotic onto one well, active microbe to other well and distilled water on

the remaining well.

5. Incubate at 37 degree centigrade for overnight in straight position.6. Then observe result.


25/61

Observation:

Result: A zone of inhibition is observed around active microbe also, it means it is producing

antibiotics to inhibit the growth.


26/61

CHAPTER : II

MOLECULAR BIOLOGY

&

BIOCHEMISTRY


27/61

Experiment no- 1

Object: To Extract the DNA from Plant Sample.

Requirement : CTAB Buffer,2% CTAB(20gm ctab),20Mm EDTA, 100mM Tris -cl pH 8(100ml Triscl stock (1M)),1.4M NaCl (280 ml NaCl stock (5M)),make upto1litre with water,Ph

7.5-8 and Autoclaved,.2% Merceptoethenol, Wash Buffer,70% Ethanol, Chloroform/IAA

(24:1), etc.

Principle:In this experiment we use gel electrophoresis . the principle for electrophoresis is

A technique used to separate and sometimes purify macromolecules especially

proteins and nucleic acid that differ in size, charge and conformation. As such , it is one of

the widely- used techniques in biochemistry and molecular biology.

Procedure:

1) Grind 1gm leaf in 5ml of preheated and Mercaptoethenol (5l) added CTAB buffer.2) Then we Transferd 500l of the homogenate in the fresh eppendorf tube .3) Incubate at 67c for 1 hrs.4) Then we Mixed gently, inversion after every 15 min during incubation.5) Then add 500l of chl: IAA (24:1).6) Then we mixed gently for inversion for 15 min.7) The spin at 10000rpm for 10 min .8) Then transfer the upper aqueous layer in the fresh eppendorf tube .9) Then we added equal volume of chilled Isopropanol.10) Then incubate over night / at 0c for 30 min.11) Then spin at 10000rpm for 10 min .12) Discard supernatant , add 300l of 70% ethanol.13) After this again spin at 10000rpm for 5 min .14)) Again discard the supernatant , air dry the pallet.15) Add 20l of T.E Buffer.


28/61

After the DNA got dissolved in TE buffer AGE was performed.

Observation : Pink Colored bands of DNA were observed when the gel was illuminated

under UV light.

Result : We got the Pink colored DNA Bands.


29/61

Experiment no- 2

Object : To isolate DNA from bacterial sample.

Requirement : : CTAB Buffer,2% CTAB(20gm ctab),20Mm EDTA, 100mM Tris -cl pH 8(100ml Triscl stock (1M)),1.4M NaCl (280 ml NaCl stock (5M)),make upto1litre with water,Ph

7.5-8 and Autoclaved,.2% Merceptoethenol, Wash Buffer,70% Ethanol, Chloroform/IAA

(24:1), etc.

Principle: In this experiment we use gel electrophoresis . the principle for electrophoresis is




Procedure:

1) Transfer 1.5ml of 24 hrs old grown bacterial culture in a fresh eppendorf tube.2) Then we spin at 5000 rpm for 5 min .3) Then we Discard Supernatant and resuspended the pallet in 467l of T.E .Buffer.4) Then we added 30l of 10 % SDS and 3l of Protenase K.5) Then incubate at 37c for 1 hrs.6) Then we add equal volume of phenol: chloform mixture in the ratio of 1:1 .7) Then mixed gently for inversion for 15 min.8) Then spin at 10000rpm for 10 min .9) Transfer the upper aqueous layer in the fresh eppendorf tube .10) At last we add 1/10 volume of 3molar potassium acetate and equal volume of chilled

isopropanol.

11) Incubate over night / at 0c for 30 min.12) Spin at 10000rpm for 10 min .13)Discard supernatant , add 300l of 70% ethanol.14)Then again spin at 10000rpm for 5 min .


30/61

15) Again discard the supernatant , air dry the pallet.16) At last add 20l of T.E Buffer.

Observation : We observed the bacterial DNA .

Result : We isolated the bacterial DNA.


31/61

Experiment no-3

Object: To Analyse a plant DNA.

Requirement : 1x TAE Buffer, ETBR , DNA ,Agarose Gel,TAE Buffer ,Sample Buffer ( Dye+DNA) etc.

Principle : Absorbance = A260/280= 1.8then DNA is Pure.

1.8 then Pritein contamination.< then lipids or carbohydrate contamination.

This is the formula used behind Quantitative Analysis.

Procedure: Qualitative Analysis

1) We take .5 ml of TAE Buffer2) Then add 24.5 ml distilled water.3) Then we add .175 gm Agarose.4) Then heated in oven for proper mixing.5) Then we left to cool and add 1l ETBR.then load in gel casting tray with comb .6) Left to solidify and then remove the comb .7) Then we insert our DNA in 1: 4 ratio in wells.8) Run the electrophoresis chamber.

Quantitive Analysis

1) We take the absorbance at 250 and 280 nm and find the quantity.2) The Absorbansce is checked as.

Observation :


32/61

Sample 260nm 280nm concentration 260/280

1 .177 .153 8850 1.15

2 .167 .135 8350 1,23

3 .139 .111 6950 1.25

4 .161 .125 8050 1.2

we observed the quality of dna bands and quantity of dna bands.

Result: We test the quality as well as quantity of plant DNA .


33/61

Experiment no: 4

Object: To isolate plasmid DNA.

Requirement: Buffer and Reagent :

P1 Buffer: Resuspension Buffer(to be stored at 4c )

50mM Tris HCl(ph-8)

10mM EDTA

100g/ml RNAse A

P2 Buffer: Lysis Buffer (room temperature)

200mM NAOH

1%SDS

P3 Buffer: Neutralization Buffer (room temp. or 4c.

3M Potassium Acetate ,pH-5.5.

Phenol (TE saturated)

Chloroform: Isoamyl alcohol (24:1)

Isoprpanol

70% ethanol

Distilled water , epindoff tubes, flask etc.

Principle: in plasmid DNA isolation we used three buffer which hilp for isolation of plasmid

DNA . first we use P1 buffer: this buffer is resuspension buffer.

Second we use P2 buffer: this buffer is called lysis buffer .used for cell lyses.

Third we use P3 buffer- this is used as Neutralization Buffer.


34/61

Procedure:

1) Transfer 1.5 ml of 24 hr old grown bacterial culture in a fresh ependorf tubes .2) Then we spin at 5000 rpm for 5 min .3) Then discard supernatant.4) Then we resuspended the pallet in 100 l of P1 buffer and 5lof RNAse.5) Then incubate at 0c for 10 min.6) Then add200l of p2 Buffer .7) Then we incubate at 0c for 10 min.8) Then add150l of p3 Buffer.9) Then incubate at 0c for 10 min.10)

Then spin at 10000 rpm for 10 min.

11)Then transfered the supernatant in the fresh ependorf tubes .12) Add equal volume of phl:chl:iaa (25:24:1) .13)Then mix gently for inversion for 15 min.14)Then spin at 10000 rpm for 10 min.15)Transfer the upper aquous layerin the fresh ependorf .16)Then add equal volume of chl:iaa (24:1).17) Then we mixed gently for inversion for 15 min.18) Then spin at 10000 rpm for 10 min.19)Then transferred the upper Aquous layer in the fresh ependorf .20)Then add 1/10volume of potassium acetate and equal volume of Isopropanol.21)Then incubate at 0c for over night .22)Then spin at 10000 rpm for 10 min.23)Then discard supernatant and add 200l of 70% Ethanol.24)Then spin at 10000 rpm for 10 min.25)Then discard supernatant and air dry the pallet and add 20 l of T.E . Buffer.

Observation: We will observe the isolated dna .

Result: The plasmid dna is isolated .


35/61

Experiment no-5

Objective :To perform Restriction Digestion .

Requirement : DNA sample, Restriction Enzyme ,R.E. Buffer ,Distilled water, Agarose,ETBR, TAE Buffer,Gel Running Buffer, Gel Casting Tray, Comb, Beaker etc.





Procedure:

1) We first take 10l of DNA sample.2) Then we add 1 l of R.E.3) Then we add 2l of R.E. buffer.4) Then we add 7l of distilled water.5) Then we incubate 37c for 1 hr.6) Then we go for Agarose Gel Electrophoresis.

Observation: We observed the Restricted Fragment.

Result: The Restricted DNA Bands are seen


36/61

Experiment no- 6

Object :To Perform Ligation.

Requirement: R DNA Insert , R Plasmid Vector , T4 DNA Ligase , T4 DNA Ligase Buffer,Distilled water , Agarose , TAE Buffer , ETBR etc.

Principle: In this experiment we use gel Electrophoresis . The Principle for Electrophoresis is

A technique used to separate and sometimes purify macromolecules especially proteins and

nucleic acid that differ in size, charge and conformation. As such , it is one of the widely-

used techniques in biochemistry and molecular biology.

Procedure:

1) First of all we take Restricted DNA insert 2l ( ECOR1) .2) Then we take Restricted Plasmid vector 2l and add those two.3) Then add T4 DNA Ligase- .5l. ( optimally act at 22c).4) Then we add DNA Ligase Buffer -1l.5) Then we add Distilled water .- 4.5l.

Total- 10l

6) Then incubate at 22c for 30 min .7) Then heat at 60c for 10 sec .8) Then we go for Agarose Gel Electrophoresis.

Observation: We observe the complete bands.

Result : Ligation is performed .


37/61

Experiment no- 7

Object :To prepare a competent cell .

Requirement : 2x LB- 500l

25% PEG ( Poly Ethylene Glycol) - 400l.

1 M Magnesium Chloride (MgCl 2) - 50l. TSS

DMSO( Dimethyl Sulphoxide) - 50l.

Principle :"Competent" cells are the one whose cell membrane has been altered in a way that

allows it to accept the incoming DNA. Naturally some competent cells have membrane proteins

which collectively helps in uptake of DNA. Artificially this can be done by making the cells

competent by treating them with Calcium Chloride which helps the foreign DNA to attach to the

cell membrane; this is then followed by a short heat shock treatment that helps the real uptake of

the DNA probably by making pores in the cell membrane

Procedure:

1) We first take 3-4 hr old grown bacterial culture. ( E.coli).2) Then take in 1.5 ml in eppendorf.3) Then spin at 5000 rpm for 5 min .4) Then we will get the pallet .5) Then add 1ml TSS ( transformation storage solution ) .6) We can store at -20c for six Month.

Observation: We observed the competent cells and mixed in TSS for storage long time.

Result : Competent cells are prepared.


38/61

Experiment no- 8

Object.:To perform Transformation of Amylase Gene containing vector in E .Coli .

Requirement : DNA , 30ml L.B. Poly Ethylene Glycol ,MgCl 2 , DMSO( Di MethylSulphoxide)

2x lb- 500l

25% PEG ( poly ethylene glycol) - 400l.

1 M Magnesium Chloride (MgCl2) - 50l. TSS

DMSO( Dimethyl Sulphoxide) - 50l.

Principle : transformation is the genetic alteration of a cellresulting from the

introduction,uptake and expression of foreign genetic material (DNA) . This is a common

laboratory technique in molecular biology.The effect was first demonstrated in 1994 by Oswald

A very.

Procedure:

1) We first take 100l Competent cells + 10l Vector.2) Then Incubate at oc for 10min.3) Then we Incubate at 42c for 145 min. heat shock4) Then we Incubate at 0c for 10 min.5) Then add 900l 2x lb .6) Then Put in shaker incubator at 37c /120 rpm for 2 hrs.7) We then got transformed E. Coli with supernatant.8) Then put for 5000 rpm for 5min.9) Then we discard the supernantent .10) We got the Transformed E.Coli.


39/61

Blue White Screening

1) We Prepared N.A Plates .2) After pouring Left to solidify.3) Then we spread 40l of X Gal in all the Plates.4) Then we spread 20l of IPTG in all the plates.5) Then we spread 5l of the Amp. In all the plates.6) And at the end we spread 20l of Transformed E.Coli .

X Gal galactosidase Blue Colored Product Blue

colonies

IPTG

X Gal White Colonies

IPTG

Observation : We observed the White Colonies .

Result: 100 % Transformation performed


40/61

Experiment no- 9

Object: To Perform Southern Blotting.

Requirement : Agarose Gel, Nitro cellulose membrane, Dry filter towels, Wet filter paper,Filter Paper Wick,.5 kg Weight, Distilled water, 1x SSC Buffer,3mm filter towel. Etc.

Principle: In this experiment we use gel electrophoresis . The Principle for Electrophoresis is




Procedure :

1) We first take the Gel containing DNA bands .2) Then we put the wick in tray .3) Then put the gel on the filter paper wick .4) Then we put the Niterocellulose membrane on the gel.5) Then we take gel sized 1mm 4wet filter paper on NCM.6) Then we put 1mm dry filter towels .7) Then we put 3mm filter towels at least 2.5 inch.8) Then at the top we put .5 kg weight .


41/61

9) Then left it for over night .10) Then we washed with distilled water .11) Then bake at 50c in hot air oven.12) Then we see the bands in UV Transalluminator.

Observation

:

We observed the DNA bands on the nitro cellulose membrane.

Result: The DNA bands are seen on Nitro Cellulose Membrane in UV Transaluminator


42/61

Experiment no- 10

Object:To Perform Polymerase Chain Reaction. {P,C,R}.

Requirement : Taq DNA Polymerase Enzyme,Taq DNA Polymerase Buffer,DNTPs,MGCL2,Primer,DNA Sample, Double Distilled Water,etc.

Principle: The purpose of a PCR (Polymerase Chain Reaction) is to make a huge number of

copies of a gene. This is necessary to have enough starting template for sequencing.PCR allows

the production of more than 10 million copies of a target DNA sequence from only a few

molecules.

Procedure: table : 1 Reaction mixture. { concentration-5u/ l}

S.NO Requirement Concentration Volume

1 Taq DNA Polymerase

Enzyme

1 unit .2 l

2 Taq DNA Polymerase

Buffer

10X 2 l

3 DNTPs, 2mM 2 l

4 MGCL2 3mM 2 l

5 Primer .5-1m 2 l

6 DNA Sample 10ng 2 l

7 Double Distilled

Water

- To make up the final

volume.


43/61

Steps:

1) Initiation: (1st denaturation) 94c- 5min2) Denaturation 94c- 30sec-2min.3) anneling ( Tm-melting temp of the primer)

Anneling temp. = Tm-5/20-40sec

4) extension 72c1min5) Repeat 30-60 cycle range of PCR .6) final extension (recovery step) 72c5 -10 min7) hold - 4c continue.

Then go for Agarose Gel Electrophoresis.

Observation : We observed thick dna bands .

Result : The DNA bands were seen .


44/61

Experiment no- 11

Object: To Perform SDS PAGE (Sodium Dodicile Sulphate Poly Acrylamide Gel

Electrophoresis).

Requirement: SDS10% , Tris hcl(8.8), D/W,APS 10%,TEMED,Tris Buffer,

Mercaptoethenol , Bromophenol Blue, CBB R250, Methanol: Glacial Acetic Acid: Distilled

water, Sucrose, etc.





Procedure: 1) Gel System.

Continuous Dis continuous

Ph -8.8, sep.gel-12.5% sep. gel-75-85%

2) Sample preparation .

Sample buffer- Tris buffer, sucrose/ glycerol, SDS , Mercaptoethenol , bromophenol

blue.


45/61

Protein sample ( equal volume add sample buffer) then boil for 5-10 min.then we cool

this sample and then load .

3) Electrophoresis

Run the sample by producing the electric field.

4) Staining :

CBBR250 - .2%

Methanol: Glacial Acetic Acid: Distilled water

( 4 : 1 : 5 )

5) Distaining solublised

Then we go for Distaining: solublised CBB dye but do not soluble CBB complex.

6 ) Agarosse Gel Electrophoresis.

Then we go for Agarose Gel Electrophoresis .


46/61

Observation: We observed the bands

.

Result: We see the separation of protein under denaturing condition


47/61

Experiment no- 12

Object :To Perform Total RNA Isolation from plant sample.

Requirement: Trizol Solution,Chloroform,70% Ethanol, Isopropanol, DEPC treated water,

By kits : Arrest extraction buffer(lysis buffer), precipitation buffer ( for protein removal) ,

Ethanol, RNA binding matrix ( bind rna ) , wash1, wash2, Elusion buffer.

Principle:

Total RNA is obtained by extracting a whole cell homgenate with phenol. The

concentrated solution of phenol disrupts hydrogen bonding in the macromolecules causing

denaturation of protein, The turbid suspension is centrifuged and two phases appear. The lower

phenol phase contains DNA and the upper aqueous phase contains carbohydrates and RNA.

Denatured protein which is present in both phases is removed by centrifugation. The RNA is

intact with alcohol added. The product this obtained is free of DNA but usually contains or

contaminated with polysaccharide. Further purification can be made by treating the preparation

with amylase.

Procedure : There are two different methods for isolation of total rna.

1}manual method.

1) We take 1gm plant sample.2) Then we mixed in 5ml of Trizol solution .3) Then transfer 500l in eppendorf .4) Then incubate at room temperature for10 min.5) Then we add equal volume of chloroform and mix gently by inversion for 15 min.6) Then Spin at 10000 rpm for 10 min.7) Then we take supernatant and add equal volume of chloroform and mix for 5 min.8) Spin at 10000 rpm for 10 min.9) Then add equal volume of Isopropanol.10)Then we Incubate at -20c for 30 min / overnight .


48/61

11)Then Spin at 10000 rpm for 10 min.12)Then discard supernatant and wash the pallet with 70% Ethanol.13)Then air dry and add 40of DEPC treated water.14) Then we go for RNA gel electrophoresis.

2}By kits ( by G . BIO SCIENCES )

1) We grind 50mg plant material in 600l of Arrest Extraction Buffer in an autoclavedmotor passcle

2) Then transferred the homogenate 600l in DEPC treated eppendorf tube .3) Then incubate at room temperature for 5 min .4) Then add 60l of precipitatory solution ( precipitation buffer).5) Then mixed by inversion for 10 min .6) Then Spin at 10000 rpm for 10 min.7) Then transfered the supernatant in the fresh appendorf tube .8) Then with mixing add 330l of ethanol and 50l of RNA binding matrix .9) Then mix by inversion and incubate at room tempe. For 5 min.10) Then spin at 10000 rpm for 5 min .( To Pallet the RNA bound to RNA binding matrix).11) Then we discard supernatant .,12)

Then resuspended the pallet in 500l of wash 1.

13) Then spin at 10000 rpm for 10 min.14) Then again discard the supernatant.15) Then resuspended the pallet in 600 l of wash 2.16) Then again spin at 10000 rpm for 10 min.17) Then discard the supernatant.18) Then again resuspended the pallet in 600l of wash 2.19) Then spin at 10000 rpm for 1 min.20) Then discard the supernatant .21) Then again resuspended the pallet in 600l of wash 2.22) Then air dried the pallet for 15 min.23) Then resuspended the pallet in 50l of pre heated 60c Ellution buffer .24) Then incubate for 5 min at 60c.


49/61

25) Then Spin at 10000 rpm for 10 min.26) Then collect the supernatant in fresh appindoff tube .27)Then go for RNA gel electrophoresis.

Observation: We observed the RNA bands with the help of white light.

Result : we got the RNA Bands.


50/61

Experiment no- 1

Object: To Extract a protein from plant leaf by Bradford Reagent .

Requirement: PEB(protein extraction buffer)

TRIS HCL - 50mM- buffering.

DTT ( DIthio theretol)- 5mM

Mgcl2-10mM

EDTA - .1mM Cell Wall Lysis

Isoascorbate-5mM

Principle: The assay is based on the ability of protein to bind commasive brilliant blue G250

and form a complex whose extinction coefficient is much greater then what of the free dye .

Procedure:

1) We take 1gm leaf.2) Then add 5ml extraction buffer.3) Then spin at 10000rpm for 10min .4) Then we take supernatant in new appindoff .5) Then again we spin at 10000 rpm for 10 min.6) Then we take supernatant .7) At end we got crudeprotein.

Bradford reagent:

1) We take Comassive Brillient blue- 5ml 95% ethanol.2) Then we add 5ml orthophosphoric acid (85%).3) Then we add 40 ml distilled water.4) Then filter it.5) Bradford is ready.


51/61

6) Now we add B.R in all the test tube -2.5 ml.Stock (BSA) G-250- mg/ml.

Working BSA1:4 ( 4- distilled water.

Observation :

Test tube Working BSA Distilled

water

Concentration

of BSA

bradford O.D.

1 0 (BLANK) 500ML 0 2.5ML 0

2 50ML 450 .02 2.5 .06

3 100 400 .04 2.5 .15

4 150 350 .06 2.5 .17

5 200 300 .08 2.5 .19

6 250 250 .1 2.5 .21

7 300 200 .12 2.5 .23

8 350 150 .14 2.5 .24

9 400 100 .16 2.5 .26

10 450 50 .18 2.5 .27

11 500 0 .2 2.5 .29

12 250(TEST) 250 .1 2.5 .21

Concentration of

Protien in Plant

sam le.


52/61

O.D

Conc. Of BSA

Result: The protein was successfully extracted.

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0

0.

02

0.

04

0.

06

0.

08

0.1

0.

12

0.

14

0.

16

0.

18

0.2

0.1

O.D.

O.D.

Test

protein

sample.


53/61

Experiment no- 2

Object:To estimate the proteins by Lowrys Method.

Requirement:

BSA (stock)- mg/ml

Working-1:4,

Reagent A -2% na2co3 in .1 N Naoh .

Reagent B-.5% cuso4 in 1%k NA tartarate.

Reagent C -50mlA+1ml B

ReagentD- F.C (1:1)

Principle: The blue color developed by the reduction of the Phosphotungstic component in

the Folin Cicolteau reagent by the amino acid Tyrocin and Tryptophan present in the protein plus

the color developed by the Burette reaction of the protein with the Alkaline Cupric Tartarate are

measured in the lowery method .

Procedure:

1) First we take 10 test tubes first blank and then in increasing order of BSAfrom 20l to 200 l.

2) Then make up the final volume200l with the help of distilled water .3) Then we add 1ml of reagent D in each test tube vortex and let stand for

10min at room temp.

4) Then we add .3ml of FC reagent and vortex the tubes immediately and letstand for 60 min at room temp.

5) Then we take absorbance at 660nm AND PLOT THE GRAPH BETWEENConc. of BSA vs Absorbance at 660nm.


54/61

Observation :

Teat Tubes BSA Distilled

Water

Conc.of

BSA

Reagent C Reagent D O.D.

1 0 1 0 5ml .5ml 0

2 .2 .8 .04 5 .5 .09

3 .4 .6 .08 5 .5 .16

4 .6 .4 .12 5 .5 .23

5 .8 .2 .16 5 .5 .33

6 1 0 .2 5 .5 .61

7 500l 500l .1 5 .5 1.44

O.D

Conc. Of BSA

Result: the Protein was estimated from plant .

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 0.04 0.08 0.12 0.16 0.2 0.1

O.D

O.D

Protein sample


55/61

Experiment no- 3

Object: To Estimate the DNA by DPA Method .

Requirement: Diphenyl Reagentdissolve 1gm of Diphenyl amine in 97.5 ml Glacial AceticAcid and then add 2.5ml of conc. Sulphuric acid . Mix well . store the reagent in brown bottle at

room temperature. DNA Stock SolutionDissolve calf thymus DNA or equivalent at .2

mg/ml in 5mM NaoH .Store at 4c.

Principle : In the presence of strong acid , the Deoxyribose moieties of DNA from

Hydroxylevulinic acid. The Hydroxylevulinic acid react with diphenylamine and produces a

blue colored complex with a lambda max of 595nm. The colored is measured at 595nm .

Procedure:

1) First we Pepped out .05ml to 1ml of DNA stock solution in to clean tubes.2) Then Make up the final volume 1 ml with distilled water.3) Then add 5ml of Diphenyl reagent to each tubes and mix well.4) Then place all the tubes in boiling water bath for 10 min and cool at room tem.5) Plot a graph using amount of DNA vs absorbance at 595 nm.

Observation:

DNA DISTILLED WATER CONC. OF DNA O.D

0 1 5ml 0.0

.2 .8 5ml .05

.4 .6 5ml .07

.6 .4 5ml .1

.8 .2 5ml .131 0 5ml .15


56/61

O.D

known conc. Of DNA

Result : So by DPA method DNA was easily extracted.

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

50l 100" 150" 200" 250" 300"

O.D.

O.D.


57/61

Experiment no-4

Object: To perform Enzyme Assay.

Requirement :

- .02 M sodium phosphate buffer, pH 6.9 with.006M Sodium Chloride .- 2zn Sodium Hydroxide .- Dinitrosalicyclic acid color reagent- prepare by dissolving 1.0 gm of 3,5-

dinitrosalicylic acid in 50ml of reagent water. Add slowly 30.00gms

sodium potassium tarterate tetrahydrate .add 20 ml of 2 N NaoH . dillite

to a final volume of 100 with reagent water.

- 1% starch . prepare by dissolving 1.0 gm soluble starch , ( Merck) in 100ml .02 M sodium phosphate buffer, pH 6.9 with.006M sodium chloride .

bring to a gentle boil to dissolve . cool and bring volume to 100 ml with

water.

- Maltose stock solution prepare by dissolving 180 mg maltose in 100ml reagent grade water in volumetric flask.

Principle : Alpha Amylases upon large polymer at internal bonds .The hydrolytic product have

alpha configuration .The activity is present in all living organism however the enzyme vary

remarkably even for tissue within a single species.

Characterstic: M.wt:51000-54000., pptimum pH 7.0 .

Procedure:

1) First we prepare Maltose .Maltose = mg / ml.

Working = 1:1

2) Then we prepare DNS . ( 100ml)1% NaoH (i.e 1gm in 100 ml )

Add 1 gmDNS ( powder )

Add .2 gmphenol crystal ( to intensify colour)


58/61

Add .05 gmNa2 sulphite (removal of organism )

Add 15 gm -KNa tartrate .

3) Starch + amylase maltose + DNS 100c ANSAO2

4) Then we take 11 test tubes and go for reaction.Observation:

Working maltose Distilled water Conc. Of maltose DNS O.D.

0 1 0 2 ML 0

.1 .9 .05 2 .01

.2 .8 .1 2 .04

.3 .7 .15 2 .06

.4 .6 .2 2 .1

.5 .5 .25 2 .12

.6 .4 .3 2 .15

.7 .3 .35 2 .17

.8 .2 .4 2 .23

.9 .1 .45 2 .26

.10 0 .5 2 .29


59/61

O.D

Conc. Of

maltose

We observed the straight line between the concentration of maltose and O.D . ( 540 nm )

Result : We check the enzyme assay.

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.05 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5

O.D.

O.D.


60/61

Experiment no- 5

Object:To check the Effect of Incubation time on enzyme activity.

Requirement: Starch , Amylase , DNS, etc.

Principle: Here in this experiment we check the effect of incubation time on Enzyme Activity .

In that case we take the graph for different time interval.

Procedure:

1) First of all we take six different test tubes .2)

Drop .5 ml starch in all those test tubes .

3) Then add .5 ml amylases and lift for incubation for 37c at different time interval.4) Then boil the test tube for 15 min .5) At last we take the O.D . for different test tubes.

Observation :

STARCH AMYLASE INCUBATE

37C

DNS BOIL O.D.

.5 ml .5 ml 0 min 2 ml Boil at100c

for 15 min

0

.5 .5 10 min 2 .16

.5 .5 15 min 2 .17

.5 .5 20 min 2 .15

.5 .5 25 min 2 .14

.5 .5 30 min 2 .11


61/61

O.D

Incubation time

So we got the max. incubation time on enzyme activity.

Result: The maximum incubation time for enzyme activity is 15 minutes.

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

0 min 10 min 15 min 20 min 25 min 30 min

O.D.

O.D.

Maximum

incubation

time

final report i

Documents