The slides tells about the basic techniques performed in biotechnology lab. a initiator should be known with these techniques so that it become easier for the one who wants to see himself in a biotechnology field.
Basic Techniques Of Biotechnology
• Isolation Of genomic DNA from Bacteria.
• Visualization of isolated DNA through Agarose gel
• Polymerase Chain Reaction (PCR).
• SDS- PAGE
• Restriction Digestion
• Nano particle synthesis & to test their antimicrobial activity.
• Plant tissue culture.
• Cultivation of Spirulina platensis by using zarrok`s media .
CONCENTRATION OF CHEMICALS
USED IN DNA EXTRACTION
• 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.
E.Coli DNA EXTRACTION
Pellet of E.coli culture is added with lysis buffer.
Lysis Buffer = TE Buffer+ SDS+ proteinase k
• After incubation add chloroform and isoamyl alcohol
instead of phenol.
• Three layers are formed:
RNA AND DNA
• After taking aqueous layer in new vial and then
centrifuge. Pellet is having RNA so take
• Add ethanol in supernatant and incubate.
• Centrifuge and take pellet.
• Air dry the pellet and store by adding TE-buffer.
STEPS OF EXTRACTION OF DNA FROM A E.coli CELL
1.5ml E.coli culture
Vortex and incubate at 37
degree centigrade for 1 hr.
Add chloroform and
Take aq.layer and
Incubate at 20 degree
cent. For 30
Take pellet and add 1ml 70%
Take pellet and air
Add TE Buffer and
• Separation of DNA,RNA,proteins by applying an electric
field to move negatively charged molecule through an
• Molecules separated in their fragments on the basis of
their size by sieving.
• It is used to
2.Separate mix population of DNA.
3.Separate RNA fragment by length.
4.Estimate size of DNA and RNA.
COMPOSITION OF AGAROSE GEL
AND BUFFER USED
• 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.
PROCESS OF GEL ELECTROPHORESIS
• 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
• 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.
ROLE OF EtBr
Role of EtBr
it is a fluorescent
dye which fluorescent after
two strands , under a
UV light. It emit a particular
wavelength of light which comes
under visible light.
POLYMERASE CHAIN REACTION OF
EXTRACTED DNA FROM E.coli
POLYMERASE CHAIN REACTION
• 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
• 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.
REQUIREMENT FOR PCR 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
TO AMPLIFY 1Kb FRAGMENT FROM EXTRACTED
TEMPLATE DNA FROM E.coli
3 step 4step
denatur annea extent final final
-ling -ion extent hold
GRAPH SHOWING PCR REACTION
BETWEEN TIME AND TEMPRETURE
PRINCIPLE OF 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
SDS-PAGE is chemically inert and produce different pore
NOTE: There is a discontinuous buffer system.
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.
STACKING GEL AND RESOLVING GEL :
2. Gel buffer
4. SDS (10%)
6. APS (20%)
CHEMICAL INGRADIENTS AND THEIR
• 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
4.Glycerol-: increase density of sample.it is non-ionic and
non-reactive toward proteins to interfering with electricity.
• 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 nonreactive ends. Hence cross linked two polyacrylamide chains
to one another results to a gel formation.
3. TEMED (Tetramethylethylene diamine)-:provide free
4. APS (Ammonium per sulfate)-:stabilize free radicals and
forms the gel.
• 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.
• Size varies as:-Cl- < glycine < SDS bound protein
NOTE: Glycine slows down in stacking gel do
move but with less mobility than Cl- ions
In resolving gel glycinate move behind Clbut ahead to proteins.
• Staining dye:- It is anionic, non-polar, nonspecifically bound to protein. Allowing
visualization of separated protein. Different
protein will appear as distinct bands within
• Distaining dye:- It is used to destain the
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
• 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
Wells formed after
(4 wells contain protein
Sample along with dye
And one well contain
Dye tracking the
Path of protein move
STAINING AND DESTAINING OF GEL
• Staining dye is used to stain the proteins
• 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.
For destaining the
shaking is provided
overnight with the
help of decoloring
INTRODUCTION OF RESTRICTION
• Restriction enzymes are enzymes isolated
from bacteria that recognize specific
sequences in DNA and then cut the DNA to
produce fragments, called restriction
• Restriction enzymes play a very important role
in the construction of recombinant DNA
molecules, as is done in gene cloning
CHEMICALS USED IN THE PROCESS
2X ASSAY BUFFER
IN VIAL 1
IN VIAL 2
• 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
INTRODUCTION OF NANOPARTICLES
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
•We can also compare it with things in the natural world.
Less than a
are up to a few
tenths of a
like these red
balls) span 1 have diameters
in the range of
DNA molecules thousands of
An ant is
A two meter tall
male is two
PROCEDURE OF EXTRACTION
• 0.01gm AgNO3 dissolved in 100ml distilled
AgNO3 dissociate after dissolving in water.
Ag+ + NO3 -
can be stabilized by adding bio
resource (having large photo
Leaves of Moringa is taken as bioresource.
Leaves were crushed and then centrifuge.
Take supernatant .
Add supernatant in .1gm AgNO3 in 100ml
• Kept onto the magnetic stirrer for overnight.
• Nanoparticles formed by changing the colour
AFTER 2 HOURS
ANTIMICROBIAL ACTIVITY OF
• Nutrient agar media was prepared for bacteria and PDA for
• 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
• Overnight incubation was given.
RESULTS SHOWING ANTIMICROBIAL
ACTIVITY OF NANOPARTICLES
• FOR BACTERIA :Species concentration of
• FOR FUNGI:
diameter of zone
Petriplates Showing Antimicrobial Activity:-
Presence of clear zone shows the inhibition of E.coli by silver
Presence of clear zone shows inhibition of pseudomonas by
Presence of clear zone around the wells indicates the inhibition of
fungi (Aspergilus niger)
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.
1st . Dot-ELISA strip +1x assay buffer+
serum sample :
+ve control zone
INCUBATE FOR 20MIN.,
2nd. Add enzyme antibody conjugate(antibody-HRP)
INCUBATE FOR 20 MIN. , WASH
No binding with
3rd . Add substrate (TMB/H2O2):
INCUBATE FOR 20 MIN. ,WASH
No binding of
Hence no blue
Spot in test zone
HRP + O2
2H2O + O2
• 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
• The production of multiples of plants is possible
in the absence of seeds or necessary pollinators
to produce seeds.
• 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.
Cultivation of Spirulina Platensis
by using of Zarrok's media
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
1- Measured 50 ml of zarrouk`s media in one flask under laminar
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
4-after adjusting OD at 0.3, Spirulina culture was put in tissue
5- suitable condition for culture was provided.
After 9 days incubation culture was at harvesting stage.