basic techniques of biotechnology
-
Upload
shalinikaushik -
Category
Technology
-
view
1.019 -
download
2
description
Transcript of basic techniques of biotechnology
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.