Basic Techniques Of Biotechnology

By:- Shalini Kaushik B.Tech (BT)4th year

CONTENTS

• Isolation Of genomic DNA from Bacteria.• Visualization of isolated DNA through Agarose gel

Electrophoresis.• Polymerase Chain Reaction (PCR).• SDS- PAGE• Restriction Digestion • Nano particle synthesis & to test their antimicrobial activity.• DOT-ELISA.• Plant tissue culture.• Cultivation of Spirulina platensis by using zarrok`s media .

EXTRACTION OF E.coli GENOMIC DNA

• TE-Buffer : 10mM tris-cl (pH- 8.0) 1mM EDTA (pH- 8.0)

• Lysis buffer (10ml):9.34ml TE-Buffer 600 µl of 10% SDS 60 µl proteinase k(20mg/ml)

Overall:: TE Buffer(20ml)= .0242gm Tris-Cl +.00744gm EDTA. Lysis Buffer(10ml)=9.34ml TE-Buffer+.06gm SDS+ 12mg proteinase k.

CONCENTRATION OF CHEMICALS USED IN DNA EXTRACTION

Pellet of E.coli culture is added with lysis buffer. Lysis Buffer = TE Buffer+ SDS+ proteinase k

E.Coli DNA EXTRACTION

LYSE THE CELL

Tris-HCl

AND EDTA

Denature the

struct-ure of protei

-n

Remo-ve out protei

n

CONT….• After incubation add chloroform and isoamyl alcohol

instead of phenol.• Three layers are formed:

AQUOS LAYER

INTERMEDIATE LAYER

BOTTOM LAYER

having

having

having

RNA AND DNA

DENATURED PROTEIN

CHLOROFORM AND ISOAMYL ALCOHOL

CONT….

• After taking aqueous layer in new vial and then centrifuge. Pellet is having RNA so take supernatant.

• Add ethanol in supernatant and incubate.• Centrifuge and take pellet.• Air dry the pellet and store by adding TE-buffer.

centrifuge

1.5ml E.coli culture

supernatant

pellet

600l lysis bufferVortex and incubate at 37

degree centigrade for 1 hr.

Add chloroform and isoamyl alcohol

PELLET

STEPS OF EXTRACTION OF DNA FROM A E.coli CELL

Take aq.layer and centrifuge

take supernatant

Add 2.5µl ethanol

Incubate at -20 degree

cent. For 30 min.

centrifuge

Take pellet and add 1ml 70% ethanol

centrifuge

Take pellet and air dry it

Add TE Buffer and incubate

ELECTROFORESIS OF ISOLATED DNA

• Separation of DNA,RNA,proteins by applying an electric field to move negatively charged molecule through an agarose matrix.

• Molecules separated in their fragments on the basis of their size by sieving.

• It is used to 1.Separate proteins. 2.Separate mix population of DNA. 3.Separate RNA fragment by length. 4.Estimate size of DNA and RNA.

INTRODUCTION

• AGAROSE GEL-:1% of 100ml is used. means .5g agarose in 49ml d.w and 1ml 50x buffer.5 µl EtBr is used.

• 600ml BUFFER-: (Tank Buffer)12ml 50x buffer + 588ml d.w.

COMPOSITION OF AGAROSE GEL AND BUFFER USED

• Prepared agarose gel at 45ºC was poured in gel casting plate where comb was already placed.

• After formation of gel, comb was removed and wells formed.

• Buffer was poured in the tank.• Sample along with dye was loaded to the well.

Marker was also added in another well.• Apply voltage and under the influence of the electric

field, movement starts.

PROCESS OF GEL ELECTROPHORESIS

Role of EtBr it is a fluorescent dye which fluorescent after intercalating between two strands , under a UV light. It emit a particular wavelength of light which comes under visible light.

ROLE OF EtBr

RESULT OF ELECTROPHORESIS OF NUCLEIC ACID

POLYMERASE CHAIN REACTION OF EXTRACTED DNA FROM E.coli

• Given by Kary mullis in 1983.• Biochemical technology in molecular biology to

amplify a single or a few copies of a piece of DNA.

• PRINCIPLE - based on DNA polymerization reaction.

• Thermal cycling consisting of repeated cycles of heating and cooling of the reaction for the DNA melting and enzymatic replication of DNA using Taq polymerase and primer sequence.

POLYMERASE CHAIN REACTION

• Nuclease free water : 18.5 µl or 37 µl• 10x Taq pol. Assay buffer : 2.5 µl or 5 µl• dNTPs : 2 µl or 1 µl• Forward primer : 2 µl or 1 µl• Reverse primer : 2 µl or 1 µl• Extracted template DNA of E.coli :1 µl or 0.5 µl• Taq polymerase : 1 µl or 0.5 µl TOTAL :: 50 µl or 25 µl

REQUIREMENT FOR PCR REACTION:

1 step 2 step 3 step 4stepInitial denatur annea extent final final Denaturation -ation -ling -ion extent hold

TO AMPLIFY 1Kb FRAGMENT FROM EXTRACTED TEMPLATE DNA FROM E.coli

94ºC

2 min.

94ºC

30sec.

60ºC

30sec.

72ºC

1min.

72ºC

2min.

4ºC

60min.

32 cycles

GRAPH SHOWING PCR REACTION BETWEEN TIME AND TEMPRETURE

TEMPRETURE

TIME

94 94

2min 30sec

60

30sec

72

1min

72

2min

4

60min

.

in ºC

REACTION INVOLVED IN PCR

.

DENATURATION

3’ 5’

5’ 3’

3’

5’ 3’

5’

ANNEALING

3’ 5’ 3’5’

5’ 5’

EXTENTION

1st CYCLE94ºC

60ºC

72ºC

.3’

3’

3’

3’ 5’

5’5’

5’

DENATURATION

3’

3’ 3’

3’

5’

5’ 5’

5’

ANNEALING

5’

5’

5’

5’3’

3’

3’

3’

5’ 5’

5’

2nd CYCLE

.EXTENTION

3’ 3’

3’

3’

3’

3’5’

5’

5’

5’ 5’

5’

5’

5’

3’ 3’

FORMATION OF 8 DNA STRANDS

RESULT OF PCR AFTER ELECTROFORESIS

Resulted DNA band of 1kb

SDS PAGE

Principle is based on the separation of protein on the basis of their size and their charge.SDS applied to protein sample to impart a negative charge linearize the protein.Electric field applied across the gel, causing the negative charged proteins to migrate across the gel towards positive electrode.SDS-PAGE is chemically inert and produce different pore size.

NOTE: There is a discontinuous buffer system.

PRINCIPLE OF SDS PAGE

Velocity of charged particles moving in electric field is

-Directly proportional to the field strength and charge on molecule-Inversely proportional to the size and viscosity of molecule.

CONT….

STACKING GEL AND RESOLVING GEL :

CHEMICALS CONCENTRATION

Stacking gel Resolving gel1. Acrylamide/ .5ml 1.98ml Bisacrylamide 2. Gel buffer 0.63ml 1.25ml (tris-HCl-:1.5M) (pH-:6.8) (pH-:8.8)3. d.water 1.32ml 1.77ml4. SDS (10%) 25 µl 50 µl 5. TEMED 1 µl 4 µl 6. APS (20%) 25 µl 38 µl

SAMPLE PREPARATION

CONT…..• ELECTRODE BUFFER :

Tris-HCl Glysin SDS 3gm 14.4gm 1gm

• SAMPLE : 30 µl protein sample + 30 µl loading dye.• MARKER : 10 µl• DYES : Staining dye distaining dye .8gm coomassie blue d.w-:630ml in 1l d.w glacial acid-:70ml methanol-:300ml

CHEMICAL INGRADIENTS AND THEIR ROLES

• Components of loading dye:-It is colorless progress through the gel. It is anionic of known electrophoric mobility. Move ahead protein.

1.Tris base-: maintain Ph. 2.BME( Beta mercapto ethanol)-:breaks disulfide bonds. 3.SDS-: linearize proteins and impart negative charge to proteins. 4.Glycerol-: increase density of sample.it is non-ionic and non-reactive toward proteins to interfering with electricity.

Cont.…..• Components of LGB and UGB buffer used:-1. Acrylamide-:When dissolved in water autopolymerization of

acrylamide takes place. Joining of molecules head to tail fashion to form single chain polymer.

2. Bisacrylamide-:Cross linking agent for polyacrylamide gel. Two acrylamide molecule coupled head to tail at their non-reactive ends. Hence cross linked two polyacrylamide chains to one another results to a gel formation.

3. TEMED (Tetramethylethylene diamine)-:provide free radicals.

4. APS (Ammonium per sulfate)-:stabilize free radicals and forms the gel.

Cont.……• Components of electrode buffer:-1. Tris-HCL-:When voltage applied. H+ ions and Cl- ions

dissociates. Cl-ve ions are highly mobile as they are small in size as well as negatively charged. Hence it is always ahead than protein and glycine.

2. SDS-:It bound to the protein.(1.4gm SDS bound 1gm protein)and form SDS bound protein complex. Coats protein with uniform negative charge.

3. Glycine-:Weak acid which is neutral or protonated in the stacking gel while it becomes glycinate or deprotonated in the resolving gel.

Cont.……

• Size varies as:-- Cl- < glycine < SDS bound protein NOTE: Glycine slows down in stacking gel do move but with less mobility than Cl- ions and protein.In resolving gel glycinate move behind Cl- but ahead to proteins.

Cont.………• Staining dye:- It is anionic, non-polar, non-

specifically bound to protein. Allowing visualization of separated protein. Different protein will appear as distinct bands within the gel.

• Distaining dye:- It is used to destain the excessive dye.

WHY STACKING GEL IS 6% AND RESOLVING GEL IS 12% ?

• Stacking gel is having less amount of acrylamide and bisacrylamide results in large pore size. Hence all proteins of different size stack in the same line ready to move from a same point.

• Resolving gel have more amount of same due to which pore size is small. So proteins distinguish acc. to different size.

PROCESS OF SDS-PAGE

• Prepared resolving gel was poured between two glass plates.

• After that stacking gel was poured over it.• Then comb was placed between the two glass

plates having gel between them.• Comb was removed and wells formed in which

protein sample with tracking dye was loaded further.

DURING PROCESS

Wells formed afterRemoving comb.(4 wells contain proteinSample along with dyeAnd one well containMarker)

Dye tracking the Path of protein move-ment.

STAINING AND DESTAINING OF GEL

• Staining dye is used to stain the proteins pathway.

• After staining remove excessive stain by destaining dye. It will left for overnight.

• After removing the excessive dye. Take the picture of bands of proteins onto the gel.

Cont.……..

For destaining the excessive dye, shaking is provided overnight with the help of decoloring shaker.

RESULTS OF SDS PAGE

RESTRICTION ENDONUCLEASE

INTRODUCTION OF RESTRICTION ENDONUCLEASE

• Restriction enzymes are enzymes isolated from bacteria that recognize specific sequences in DNA and then cut the DNA to produce fragments, called restriction fragments.

• Restriction enzymes play a very important role in the construction of recombinant DNA molecules, as is done in gene cloning experiments.

CHEMICALS USED IN THE PROCESS

CHEMICALS CONCENTRATION IN VIAL 1

CONCENTRATION IN VIAL 2

2X ASSAY BUFFER 12.5µl 12.5µl

DNA 10µl 10µl

Eco.R1 1.5µl _

Hind III _ 1.5µl

PROCEDURE

• Add these chemicals in two different vials.• Then we started electrophoresis.• In four different wells, we add In 1st well DNA digested with EcoR1(vial1) In 2nd well DNA digested with Hind III(vial2) In 3rd well standard DNA (undigested DNA) In 4th well digested DNA

RESULTS

NANOPARTICLESFROM BIORESOURCES

INTRODUCTION OF NANOPARTICLES

Less than a nanometerIndividual atoms

are up to a few tenths of a

nanometer in diameter

NanometerTen shoulder- to-shoulder hydrogen

atoms (blue balls) span 1 nanometer.

DNA molecules are about

2.5nm wide

Thousands of nanometers

Biological cells, like these red blood cells, have diameters in the range of thousands of nanometers

A million nanometers

An ant  is millions of

nanometers across

Billions of nanometers

A two meter tall male is two

billion nanometers

tall

A nanometer is a billionth of a meter or 10-9 m.How small is nanometer?•If a baseball is the size of Earth, a nanoparticle would be the size of an apple.•We can also compare it with things in the natural world.

PROCEDURE OF EXTRACTION

• 0.01gm AgNO3 dissolved in 100ml distilled water .

AgNO3 dissociate after dissolving in water. AgNO3 Ag+ + NO3

-

can be stabilized by adding bio resource (having large photo -synthetic activity)

Cont……..

• Leaves of Moringa is taken as bioresource. • Leaves were crushed and then centrifuge.• Take supernatant .• Add supernatant in .1gm AgNO3 in 100ml

distilled water.• Kept onto the magnetic stirrer for overnight.• Nanoparticles formed by changing the colour

of solution.

Cont………

.AFTER 2 HOURS

AFTER OVERNIGHT STIRRING

ANTIMICROBIAL ACTIVITY OF NANOPARTICLESPROCEDURE -:• Nutrient agar media was prepared for bacteria and PDA for

fungi.• Media was autoclaved and poured in the petriplates. In

NA, some plates was inoculated E.coli and some was inoculated with pseudomonas.

• While in PDA, A.niger was inoculated.• 5 wells were made in a single plate in which 20µl,40µl,60µl,

80µl of nanoparticles were poured and ketoconazole was added in middle well in PDA plate and streptomycin in NA plates.

• Overnight incubation was given.

RESULTS SHOWING ANTIMICROBIAL ACTIVITY OF NANOPARTICLES

• FOR BACTERIA :- Species concentration of diameter of nanoparticles inhibition zone E.coli 20 µl 9mm 40 µl 10mm 60 µl 11mm 80 µl 13mm

Cont.……….

pseudomonas 20 µl 9mm 40 µl 10.5mm 60 µl 11mm 80 µl 12.5mm• FOR FUNGI: Sps. Conc. of diameter of zone nanoparticles of inhibition A.niger 20 µl 7mm 40 µl 9.5mm 60 µl 10mm 80 µl 11mm

RESULTS

Petriplates Showing Antimicrobial Activity:-

Presence of clear zone shows the inhibition of E.coli by silver nanoparticles

Cont……..

Presence of clear zone shows inhibition of pseudomonas by nanoparticles

Cont………..

Presence of clear zone around the wells indicates the inhibition of fungi (Aspergilus niger)

Dot – ELISAENZYME LINKED

IMMUNOSORBENT ASSAY

INTRODUCTION OF Dot - ELISA

• Antigen is directly sandwiched between two antibodies which react with two different epitopes on the same antigen.

• One of the antibodies is immobilized onto the solid support and second is linked to the enzyme.

• Antigen present in the test sample is first linked to the immobilized antibody and then with the enzyme linked antibody.

• Incubate the strip with an appropriate chromogenic substrate which is converted into colored and insoluble product.

.

DIAGRAM SHOWING THE PROCEDURE OF ELISA

PROCEDURE

1st . Dot-ELISA strip +1x assay buffer+ serum sample : sequence A sequence B-ve control Test zone+ve control zone INCUBATE FOR 20MIN., WASH sequence A sequence BNo binding strip(Ab)as specific +antigen is absent samplein sample (Ag)

Cont.……….

2nd. Add enzyme antibody conjugate(antibody-HRP)

INCUBATE FOR 20 MIN. , WASH sequence A sequence B

No binding with Ab-HRP+

enzyme linked (Ag+strip(Ab)) antibody

Cont.…………….

3rd . Add substrate (TMB/H2O2):INCUBATE FOR 20 MIN. ,WASH

Sequence A Sequence B No binding of Binding of the substrate. Substrate Hence no blue cause blueSpot in test zone spot.

therefore, 2H2O2 2H2O + O2

HRP + O2 BLUE COLOUR

TMB

PLANT TISSUE CULTURE

INTRODUCTION

• Plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues or organs under sterile conditions on a nutrient culture medium of known composition.

• Plant tissue culture is widely used to produce clones of a plant in a method known as micro propagation.

• The production of multiples of plants is possible in the absence of seeds or necessary pollinators to produce seeds.

PROCEDURE

• Nutritional medium was prepared by the addition of some macronutrients, micronutrients, vitamins& organics for the inoculation of seed for callus culture(pH-5.7).

• Moong seeds are washed with the tap water for 2 times.• Now washed with the detergent.• Again washed with the tap water twice.• Now washed with the distilled water twice.• Then washed with the .1% HgCl2 for 1 min.• Now washed with the autoclaved distilled water for three times.• Now inoculate the seeds into the MS medium.• Flasks were kept in the growth chamber for proper growth.

RESULTS OF PLANT TISSUE CULTURE

Cultivation of Spirulina Platensis by using of Zarrok's media

INTRODUCTION

SPIRULINA is a microscopic blue green algae in the shape of a spiral coil living in sea & fresh water. spirulina is the common name for human & animal food produced from two species of cynobacteria ; arthrospira platensis and arthrospira .though referred to as algae because they are aquatic organisms capable of photosynthesis. cynobacteria are not related to any of the various eukaryotic algae.

Composition of Zarrok’s Media NaHCo3 - 4.8gm/lt

NaNo3 - 2.5gm/lt

NaCl - 1gm/lt K2So4 - 1gm/lt

K2HPo4 - 0.5gm/lt

MgSo4.7H2O- 0.2gm/lt

FeSo4.7H2O- 0.01gm/lt

CaCl2.2H2O - 0.04gm/lt

EDTA- 0.08gm/lt

PROCEDURE1- Measured 50 ml of zarrouk`s media in one flask under laminar air flow.

2- culture of Spirulina was inoculated Platensis in 50 ml of zarrouk’s media under laminar .

3 - OD up to 0.3 by using spectrophotometer at 750 nm was adjusted.

4-after adjusting OD at 0.3, Spirulina culture was put in tissue culture laboratory .5- suitable condition for culture was provided.

RESULTSAfter 9 days incubation culture was at harvesting stage.