1.
Basic Techniques Of Biotechnology
By:-
Shalini Kaushik
B.Tech (BT)
4th year

2.
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 .

3.
EXTRACTION OF E.coli GENOMIC
DNA

4.
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.

5.
E.Coli DNA EXTRACTION
Pellet of E.coli culture is added with lysis buffer.
Lysis Buffer = TE Buffer+ SDS+ proteinase k
LYSE THE
CELL
TrisHCl
AND
EDTA
Denat
ure
the
structure of
protei
-n
Remove out
protei
n

6.
CONT….
• After incubation add chloroform and isoamyl alcohol
instead of phenol.
• Three layers are formed:
AQUOS LAYER
INTERMEDIATE
LAYER
BOTTOM
LAYER
having
RNA AND DNA
having
DENATURED PROTEIN
having
CHLOROFORM AND
ISOAMYL ALCOHOL

7.
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.

8.
STEPS OF EXTRACTION OF DNA FROM A E.coli CELL
centrifuge
supernatant
pellet
1.5ml E.coli culture
Vortex and incubate at 37
degree centigrade for 1 hr.
600l lysis
buffer
PELLET
Add chloroform and
isoamyl alcohol

9.
Take aq.layer and
centrifuge
Incubate at 20 degree
cent. For 30
min.
centrifuge
take supernatant
Add 2.5µl
ethanol

10.
Take pellet and add 1ml 70%
ethanol
centrifuge
Take pellet and air
dry it
Add TE Buffer and
incubate

11.
ELECTROFORESIS OF ISOLATED
DNA

12.
INTRODUCTION
• 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.

13.
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.

14.
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
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.

15.
ROLE OF EtBr
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.

16.
RESULT OF ELECTROPHORESIS OF NUCLEIC
ACID

17.
POLYMERASE CHAIN REACTION OF
EXTRACTED DNA FROM E.coli

18.
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
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.

19.
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

20.
TO AMPLIFY 1Kb FRAGMENT FROM EXTRACTED
TEMPLATE DNA FROM E.coli
1 step
2 step
3 step 4step
Initial
denatur annea extent final final
Denaturation -ation
-ling -ion extent hold
94ºC
94ºC
60ºC
72ºC
72ºC
4ºC
2 min.
30sec.
30sec.
1min.
2min.
60min.
32 cycles

21.
GRAPH SHOWING PCR REACTION
BETWEEN TIME AND TEMPRETURE
.T
E
M
P
R
E
T
U
R
E
94
2min
94
30sec
72
72
1min
2min
in
ºC
60
30se
c
4
60min
TIME

22.
REACTION INVOLVED IN PCR
3’
5’
.
1st
5’
3’
CYCLE
DENATURATION
3’
94ºC
5’
5’
3’
60ºC
ANNEALING
5’
3’
3’
5’
5’
5’
EXTENTION
72ºC

23.
3’
5’
.
5’
5’
3’
3’
3’
5’
DENATURATION
5’
3’
5’
3’
5’
2nd CYCLE
3’
3’
5’
ANNEALING
3’
5’
3’
5’
5’
5’
5’
5’
3’
3’
5’

24.
.
EXTENTION
3’
5’
3’
5’
3’
5’
5’
3’
3’
5’
5’
5’
3’
3’
FORMATION OF 8 DNA STRANDS
3’
5’

25.
RESULT OF PCR AFTER ELECTROFORESIS
Resulted DNA band of 1kb

26.
SDS PAGE

27.
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
positive electrode.
SDS-PAGE is chemically inert and produce different pore
size.
NOTE: There is a discontinuous buffer system.

28.
CONT….
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.

29.
SAMPLE PREPARATION
STACKING GEL AND RESOLVING GEL :
CHEMICALS
1. Acrylamide/
Bisacrylamide
2. Gel buffer
(tris-HCl-:1.5M)
3. d.water
4. SDS (10%)
5. TEMED
6. APS (20%)
CONCENTRATION
Stacking gel
.5ml
0.63ml
(pH-:6.8)
1.32ml
25 µl
1 µl
25 µl
Resolving gel
1.98ml
1.25ml
(pH-:8.8)
1.77ml
50 µl
4 µl
38 µl

30.
CONT…..
• ELECTRODE BUFFER :
Tris-HCl
3gm
Glysin
14.4gm
SDS
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

31.
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.

32.
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 nonreactive 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.

33.
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.

34.
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 Clbut ahead to proteins.

35.
Cont.………
• Staining dye:- It is anionic, non-polar, nonspecifically 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.

36.
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.

37.
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.

38.
DURING PROCESS
Wells formed after
Removing comb.
(4 wells contain protein
Sample along with dye
And one well contain
Marker)
Dye tracking the
Path of protein move
-ment.

39.
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.

40.
Cont.……..
For destaining the
excessive dye,
shaking is provided
overnight with the
help of decoloring
shaker.

41.
RESULTS OF SDS PAGE

42.
RESTRICTION
ENDONUCLEASE

43.
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.

44.
CHEMICALS USED IN THE PROCESS
CHEMICALS
2X ASSAY BUFFER
CONCENTRATION
IN VIAL 1
12.5µl
CONCENTRATION
IN VIAL 2
12.5µl
DNA
10µl
10µl
Eco.R1
1.5µl
_
_
1.5µl
Hind III

45.
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

46.
RESULTS

47.
NANOPARTICLES
FROM BIORESOURCES

48.
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
apple.
•We can also compare it with things in the natural world.
Less than a
nanometerIndi
vidual atoms
are up to a few
tenths of a
nanometer in
diameter
Nanometer
Thousands of
nanometers
Ten shoulderto-shoulder
Biological cells,
hydrogen
like these red
atoms (blue
blood cells,
balls) span 1 have diameters
nanometer.
in the range of
DNA molecules thousands of
nanometers
are about
2.5nm wide
A million
nanometers
An ant is
millions of
nanometers
across
Billions of
nanometers
A two meter tall
male is two
billion
nanometers
tall

49.
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)

50.
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.

51.
Cont………
.
AFTER 2 HOURS
AFTER OVERNIGHT
STIRRING

52.
ANTIMICROBIAL ACTIVITY OF
NANOPARTICLES
PROCEDURE -:
• 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.

53.
RESULTS SHOWING ANTIMICROBIAL
ACTIVITY OF NANOPARTICLES
• FOR BACTERIA :Species concentration of
nanoparticles
E.coli
20 µl
40 µl
60 µl
80 µl
diameter of
inhibition zone
9mm
10mm
11mm
13mm

54.
Cont.……….
pseudomonas
20 µl
40 µl
60 µl
80 µl
• FOR FUNGI:
Sps.
Conc. of
nanoparticles
A.niger
20 µl
40 µl
60 µl
80 µl
9mm
10.5mm
11mm
12.5mm
diameter of zone
of inhibition
7mm
9.5mm
10mm
11mm

55.
RESULTS
Petriplates Showing Antimicrobial Activity:-
Presence of clear zone shows the inhibition of E.coli by silver
nanoparticles

56.
Cont……..
Presence of clear zone shows inhibition of pseudomonas by
nanoparticles

57.
Cont……….
.
Presence of clear zone around the wells indicates the inhibition of
fungi (Aspergilus niger)

58.
Dot – ELISA
ENZYME LINKED
IMMUNOSORBENT ASSAY

59.
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.

60.
DIAGRAM SHOWING THE PROCEDURE OF ELISA
.

61.
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 B
No binding

62.
Cont.……….
2nd. Add enzyme antibody conjugate(antibody-HRP)
INCUBATE FOR 20 MIN. , WASH
sequence A
sequence B
No binding with
enzyme linked
antibody
Ab-HRP+
(Ag+strip(Ab))

63.
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 blue
Spot in test zone
spot.
therefore,
2H2O2
HRP + O2
TMB
2H2O + O2
BLUE COLOUR

64.
PLANT TISSUE
CULTURE

65.
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.

66.
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.

67.
RESULTS OF PLANT TISSUE CULTURE

68.
Cultivation of Spirulina Platensis
by using of Zarrok's media

69.
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.

70.
Composition of Zarrok’s Media
NaHCo3 NaNo3 NaCl K2So4 K2HPo4 MgSo4.7H2OFeSo4.7H2OCaCl2.2H2O EDTA-
4.8gm/lt
2.5gm/lt
1gm/lt
1gm/lt
0.5gm/lt
0.2gm/lt
0.01gm/lt
0.04gm/lt
0.08gm/lt

71.
PROCEDURE
1- 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.

72.
RESULTS
After 9 days incubation culture was at harvesting stage.