protocols of bio degradation
TRANSCRIPT
-
8/4/2019 Protocols of Bio Degradation
1/26
3. Materials and Methods
3.1 Collection of Soil Samples for Bacterial Strains Isolation
The soil samples containing the bacteria were collected from different areas of Tadaepalli
and Mangalagiri, Guntur District of Andhra Pradesh where there was an accumulation of
the petroleum spills for Lead deposits and where as for Mercury degradation samples
were collected from Seashore of Suryalanka, Baptla, Guntur District, Andhra Pradesh.
The soil sample was collected from a depth of 15 inches depth in to the soil where there
was high moisture content, because the Bacteria need moisture content for growth. The
soil sample collected was dispensed in the sterile bags and sealed and the sample was
brought to the laboratory.
3.2 Screening of Lead and Mercury Degrading Bacteria
Soil samples were collected from the sites where lead and Mercury effluents were
discharged. Different media like Nutrient Agar, Lauria Bertani agar plates containing
Lead and Mercury to a final concentration of 5.0ppm and 7.5ppm respectively. Ten fold
serial dilutions of the soil samples were made in distilled water and 0.1ml of 10 -5 to 10-9
dilutions were plated on different media. The plates were incubated at 37C for 24 hours
and observed for clearance of degradation surrounding the colonies after incubation. The
colonies, which cleared the degradation, were transferred to similar agar plates containing
5.0 ppm of Pbcl2 for single colony isolation of Lead degrading organism. Where as in
another set of plates 7.5 ppm concentration of HgCl2 is used for Single colony isolation of
Mercury degrading organism Isolation. The isolated colonies were streaked on Nutrient
-
8/4/2019 Protocols of Bio Degradation
2/26
agar slants for growth and maintenance. Stock cultures were sub cultured monthly and
stored at 4C. The cultures were confirmed based on morphology by Grams staining.
Media and culture conditions
The medium used for the maintenance of the isolated fungal strain was Potato Dextrose
agar medium with the following ingredients:
Composition
Ingredients Amount
Peptone 5g
Beaf Extract 3g
Sodium Chloride 3g
Agar 2%
Distilled water 1000ml
Final pH 7.2-+0.2
a. The above ingredients were weighed and dissolved in distilled water in a conical
flask.
b. The pH of the medium was adjusted by adding acid or alkali.
c. The conical flask containing the medium was autoclaved at 1210c at 15 lbs pressure
for 15 min.
d. 15ml of the medium was dispensed in to sterile petriplates and allowed to solidify.
-
8/4/2019 Protocols of Bio Degradation
3/26
The Bacterial cultures were streaked on Nutrient Agar slants. Stock cultures were sub
cultured monthly and stored at 4C.
3.3 Identification of the Bacteria
The isolated bacterium was identified using different staining techniques,
different biochemical and molecular techniques.
3.3.1 Simple Staining
Bacteria are very small and transparent when observed with a wet mount
preparation. In order to observe their cell characteristics, they need to be stained
(Dyed). This method consists of preparing a smear that is air dried and heat fixed
and adding a stain to the bacteria on the slide. The simple stain consists of one
dye. The dye adheres to the cell wall and colors the cell making it easier to see.
Basic stains, such as methylene blue, Gram saffranin or Gram crystal violet are
used for staining the most bacteria. These stains will readily give up a hydroxide
ion or accept a hydrogen ion, which leaves the stain positively charged. Since the
surface of most bacterial cells is negatively charged, these positively charged
stains adhere readily to the cell surface.
Materials
Heat-fixed bacterial smears.
Methylene blue, Crystal violet, or Saffranin to act as simple stain.
Ppaper towels.
-
8/4/2019 Protocols of Bio Degradation
4/26
Microscope.
Procedure
A small drop of water was placed on a slide with a toothpick making the
culture to be in contact with water and air dried.
The dried slide was placed on the staining rack and flooded for 1 minute
with the stain.
The slide was rinsed with tap water, tilting the slide slightly to washout all
the stain from the slide by gently tapping.
The slide was placed on the bibulous paper and blot dried.
The slide was examined under the microscope and the results were
recorded.
3.3.2 Gram Staining
Several different kinds of bacteria will be examined by gram staining, a method
developed by Christian Gram in 1884 for categorizing bacteria on the basis of
differences in cell wall structure. Gram positive (+) bacteria stain a dark purple
color, while gram negative (-) bacteria stain a light red.
Microscopy reveals at least two different types of cell wall structure: single and
double. The walls of gram +ve bacteria consist of a single, thick, continuous
layer, whereas those of gram ve bacteria consist of at least two readily
-
8/4/2019 Protocols of Bio Degradation
5/26
distinguishable layers, each considerably thinner than the wall of a gram +ve
bacterium. In addition, chemical analyses reveal substantial differences in the
chemical composition of the two kinds of cell walls. In preparing a gram stain, the
first step stains both kinds of cells, gram +ve and gram ve, with a deep purple
dye complex. The next steps expose the cells to a decolorizing agent such as
alcohol and then to the counterstaining red dye Saffranin O. Since the cell wall of
the gram +ve bacterium constitutes a barrier to the decolorizing agent, the gram
+ve bacterium remains purple, while the gram -ve bacterium is decolorized and
appears light red due to the color of the red
Materials and Reagents
24 h old culture
Inoculating loops
Blotting papers
Bunsen burner and
Microscope
Crystal Violet (Huckers)
Solution A
Crystal Violet (90% dye content)
2 g
-
8/4/2019 Protocols of Bio Degradation
6/26
Ethyl alcohol (95%)
20 ml
Solution B
Ammonium Oxalate
0.8 g
Distilled water
80 ml
Dissolve the crystal Violet in ethy alcohol an dthe ammonium oxalate in
distilled water, Mix solutions A and B
Grams Iodine
Iodine 1 g
Potassium iodide 2 g
Distilled water 300 ml
Dissolve iodine and potassium iodide in distilled water
Ethyl alcohol (95%)
-
8/4/2019 Protocols of Bio Degradation
7/26
Ethyl alcohol (100%) 95 ml
Distilled water 5 ml
Safranin (2.55 solution in 95 % ethyl alcohol) 10 ml
Distilled water 100 ml
Procedure for gram staining
A small drop of water was placed on a slide with a
toothpick making the culture to be in contact with water and air
dried.
The dry slide was passed through a Bunsen burner flame
for 2 to 3 minutes to heat fix the smear.
The slide was placed on a staining rack and the slide was
flooded with crystal violet for 2 minutes.
The slide was rinsed with water from a squirt bottle.
The slide was flooded with grams iodine for 1 minute and
rinsed with water.
Then the slide was subjected to decolonization step with
95% ethanol by adding 20 drops of 95% ethanol to the slide and
the slide was left for 10 seconds and then rinsed with water from a
-
8/4/2019 Protocols of Bio Degradation
8/26
squirt bottle. Only gram + bacterial will retain the deep purple dye.
The gram bacteria will be colorless at thwas point.
Then the slide was counterstained with Saffranin for 60
seconds. This was carried out to visualize materials that have not
been stained by the deep purple crystal violet.
Then the slide was rinsed with water from a squirt bottle
and blot dried.
The slide was observed under the microscope (Olympus
Inverted microscope) first with low power objective and the slide
was placed under immersion oil directly on the base of the
microscope and it was examined with an oil immersion objective.
3.4 Biochemical Tests
3.4 Biochemical Tests
3. 4.1 Indole production test
Tryptophan an essential amino acid is oxidized by some bacteria by the
tryptophanase resulting in the formation of indole, pyruvic acid and ammonia.
The indole test is performed by inoculating a bacterium into tryptone broth, the
indole produced during the reaction is detected by adding Kovacs reagent
(Dimethyl amino benzaldehyde) which produces a cherry-red reagent layer.
Requirements
Nutrient broth cultures
-
8/4/2019 Protocols of Bio Degradation
9/26
Tubes containing 1% Tryptone broth, 5 ml/tube
Kovacs reagent
Dropper bottle
1 ml pipette
Bunsen burner
Inoculating needle
Preparation of tryptone broth (pH 7.0)
Tryptone 10 g
Calcium chloride 0.01-0.03 M
Sodium chloride 5 g
Distilled water 1000 ml
Procedure
Tryptone broth was prepared as per the composition.
Tryptone broth tubes were inoculated with the cultures and keep
one tube was kept as control.
The inoculated tubes were incubated at 350C for 48 h.
After 48 h of incubation, I ml of Kovacs reagent was added to
each tube including control.
Shake the tubes to stand to permit the reagent to come to the top.
-
8/4/2019 Protocols of Bio Degradation
10/26
3.4.2 Methyl red and Voges proskauer tests
The methyl red test and VP test are used to distinguish between bacteria that
produce large amount of acid and those that produce the neutral product acetoin
as an end product. Both these are performed simultaneously because they are
physiologically related and are performed on the same medium MR_VP broth. In
these tests, if an organism produces large amount of organic acids (end products)
from glucose the medium will remain red (a positive test) after the addition of
methyl red a pHindicator (pH
-
8/4/2019 Protocols of Bio Degradation
11/26
Bunsen burner
Inoculating loop
Preparation of MRVP broth (pH6.9) tubes
Peptone 7 g
Dextrose/Glucose 5 g
Potassium phosphate 5 g
Distilled water 10000 ml
Procedure
5 ml broth was poured in each test tube and sterilized by autoclave
at 15 lbs pressure for 15 minutes.
MRVP tubes were inoculated with test cultures and one tube was
kept as control.
The inoculated tubes were incubated at 350C for 48 h.
5 drops of methyl red indicator was added to the tube of each set.
12 drops of V-P reagent was added to the test tubes.
The tubes were shaken gently and the caps were off to expose the
media to oxygen.
-
8/4/2019 Protocols of Bio Degradation
12/26
The reaction was allowed to complete for 15-30 minutes.
3.4.3 Gelatin Hydrolysis (Production of Gelatinase)
Gelatin is a protein produced by hydrolysis of collagen a major component of the
connective tissue and tendons in humans and other animals, Hydrolysis of gelatin
is brought about by microorganisms capable of producing proteolytic exoenzyme
known as gelatinase, which acts to hydrolyze this protein to amino acids,
Hydrolysis of gelatin can be identified by growing the microorganisms in the
nutrient gelatin. If the degradation of gelatin occurs in the medium by an
exoenzyme produced by the bacteria , it can be detected by lequification by
flooding the plates with the protein precipitating material (By adding the gelatin
plates with mercuric chloride solution and observing for the clearing around the
line of growth).
Materials required
24 h culture
Gelatin agar medium (Hi Media)
Mercuric chloride solution
Sterile petri dishes
Inoculating loops
Procedure
-
8/4/2019 Protocols of Bio Degradation
13/26
The gelatin agar medium was prepared according to standard
protocol and sterilized at 121C for 15 minutes and cooled to 45 to 50C.
The sterilized medium was poured in to sterile petri dishes and
allowed to solidify.
The culture was inoculated by streaking on to the gelatin agar
plates.
The plates were incubated at 37 C for 4 to 7 days.
The plates after incubation period were flooded with mercuric
chloride solution and the plates were allowed to stand for 5 to 10 minutes.
3.4.4 Starch Hydrolysis
Amylase is an exoenzyme produced by the bacteria the hydrolysis starch a
polysaccharide, the ability of the bacteria to produce amylase is determined by
adding a indicator iodine solution starch in the presence of iodine produces dark
blue coloration of the medium and an yellow zone around the colony which
indicate the amylolytic activity
Requirements
24 h old culture.
Starch Agar Medium (Hi Media).
Grams iodine solution.
Sterile petri dishes.
Dropper.
-
8/4/2019 Protocols of Bio Degradation
14/26
Inoculating loop.
Procedure
The starch agar medium was prepared according standard protocol
and sterilized at 121C for 15 minutes and the medium was cooled to
45C.
The sterilized medium was poured in to sterile petri dishes and
allowed to solidify.
Each of the starch plate was marked with the name of the organism
The culture was inoculated by streaking on to the starch agar plates
The plates were incubated at 37 C for 48 hours in inverted
position.
The incubated plates were flooded with iodine solution with a
dropper for 30 seconds.
The excess iodine solution was poured off.
The plates were examined for the colour change in the medium
after the addition of the iodine solution which indicates the hydrolysis of
starch.
3.4.5 Oxidase Production
The production of oxidase is one of the most significant tests we have for
differentiating certain groups of bacteria. For example, all the Enterobacteriaceae
are oxidase-negative and most species ofPseudomonas are oxidase-positive.
-
8/4/2019 Protocols of Bio Degradation
15/26
Another important group, the Neisseria, are oxidase producers. This test: The
first method utilizes the entire TSA plate; the second method is less demanding in
that only a loopful of organisms from the plate is used. Both methods are equally
reliable.
Oxidase positive bacteria possess cytochrome oxidase or indophenol oxidase (An
iron containing haeme protein). These both catalyse the transport of electrons
from donor compounds (NADH) to electron acceptors (usually oxygen). The test
reagent, N, N, N, N-tetra-methyl-p-phenylenediamine dihydrochlorideacts as an
artificial electron acceptor for the enzyme oxidase. The oxidised reagent forms the
coloured compound indophenol blue. The cytochrome system is usually only
present in aerobic organisms which are capable of utilizing oxygen as the final
hydrogen receptor. The end product of this metabolism is either water or
hydrogen peroxide (Broken down by catalase).
Requirements
24 h old culture.
Nutrient Agar Plates
N, N, N, N-tetra-methyl-p-phenylenediamine dihydrochloride
Bunsen burner.
Inoculating loop.
-
8/4/2019 Protocols of Bio Degradation
16/26
The medium Nutrient Agar (pH 7) plates was prepared according
to the stranded procedure and the medium was sterilized at 121C for 15
minutes.
The Nutrient Agar plates were inoculated with the culture.
The inoculated plates were incubated at 35C for 24 to 48 h.
Pour a few drops of a freshly prepared solution of 1%N, N, N, N-
tetra-methyl-p-phenylenediamine dihydrochloride over the colony to be
tested. Colonies which change to a deep blue colour indicate a positive
reaction.
3.4.6 Catalase Activity
During aerobic process that take place in the presence of oxygen in the bacterial
cell, H2O2 is produced which is lethal to the cell. The enzyme catalase is also
produced by the bacteria that produced H2O2. The enzyme catalase breaks down
the hydrogen peroxide to water and oxygen and helps the bacteria to survive.
Catalase
2 H2O2 -------------- 2H2O + O2
-
8/4/2019 Protocols of Bio Degradation
17/26
This test was performed by adding H2O2 to Tripticase soya agar plates. The
addition of H2O2 results in formation of the bubbles which indicate the release of
the oxygen which mean the test is positive
Requirements
24 h old culture.
Trypticase soya agar plates.
Hydrogen peroxide (3%).
Bunsen burner.
Inoculating loop.
Trypticase Soya Agar composition
Trypticase 15 g (Animal peptone)
Phytone 5 g ( Soyapeptone)
Sodium Chloride 5 g
Agar 15 g
Make upto 1000 ml with Distilled water
Procedure
-
8/4/2019 Protocols of Bio Degradation
18/26
The medium Trypticase soya agar (pH 7.3) plates was prepared
according to the stranded procedure and the medium was sterilized at
121C for 15 minutes.
The Trypticase soya agar plates were inoculated with the culture.
The inoculated plates were incubated at 35C for 24 to 48 h.
The inoculated plates were observed for the presence or absence of
the gas bubble.
The formation of the gas bubbles on addition of the H2O2 indicate
the release of free oxygen which indicate the test is positive for production
of catalse by the bacteria
Utilization of macro-nutrients by Lead and Mercury Degrading isolates
The basal medium contained
Glucose, 10 g
NaN03, 2 g
MgS04.7H20, 0.2 g
CaCl2, 0.3 g
Thiamine hydrochloride, 0.5 mg
Distilled water-1000 ml
Procedure
-
8/4/2019 Protocols of Bio Degradation
19/26
The ingredients of basal medium were weighed and dissolved in distilled water in a
conical flask.
The pH of the medium was adjusted to 5.5 by adding either acid or alkali.
Then the conical flask containing medium was autoclaved at 15 lbs pressure for 15
minutes at 121OC
Antibiotic (Streptomycin) was added to the medium after cooling and mixed gently.
Then the medium was inoculated with the loop ofPleurotus isolates.
Each macro-element to be used, one having all the macro -nutrients and the other having
none.
Then the flasks were kept at room temperature for growth observation.
3.10. Effect of trace elements on the degradation of Lead and Mercury isolates
The ingredients of basal medium were weighed and dissolved in distilled water in a
conical flask.
The pH of the medium was adjusted to 5.5 by adding either acid or alkali.
Then the conical flask containing medium was autoclaved at 15 lbs pressure for 15
minutes at 121OC
Antibiotic (Streptomycin) was added to the medium after cooling and mixed gently.
Then the medium was inoculated with the loop ofPleurotus isolates.
Five trace elements (copper, iron, manganese, cobalt and zinc in their sulphate form),
were added separately to the basal medium at the rate of 10 mg/1000 cm3.
Two sets of controls were also prepared. The first consisted of all the trace elements
while the second set contained basal medium without any micro-element.
-
8/4/2019 Protocols of Bio Degradation
20/26
Then the flasks were kept at room temperature for growth observation.
Isolation of DNA from Mercury and Lead Degrading Isolates
The DNA from Mercury and Lead degrading isolates were prepared as per the protocol
1.5ml of overnight culture of each isolate was taken into different 1.5ml centrifuge tubes,
Centrifuged at 8000rpm for 5min.
Supernatant was discarded and briefly vortex pellet to dislodge.
400l of Lysis buffer [1.5M NaCl, 10mM Tris-HCl (pH8.0), 5% SDS] was added to the
dislodge pellet and incubated in water bath for 15min at 65C.
The solution was cooled to room temperature and 130l of potassium acetate (pH5.2) was
added and mixed. The solution was incubated for 5 minutes in room temperature.
The sample was centrifuged at 10000rpm for 10min.Clear supernatant was transferred into
fresh 1.5ml tube. Equal volume of isopropanol was added, gently inverted for 5 times and
incubated 10min in room temperature.
The sample was centrifuged at 10000rpm for 10min. Supernatant was discarded and pellet
is washed with 75% ethanol.
The pellet was dried in room temperature for another 15 minutes. Pellet was dissolved in
100l of TE buffer. To remove RNA from the preparation, 10l of RNase A (10mg/ml) was added
and incubated at 37C for 1 hour.
3.12.4 Purification of Isolates DNA
Solutions
-
8/4/2019 Protocols of Bio Degradation
21/26
RNase A: RNase A of 10 mg/ml was dissolved in 10 mM Tris-HCl (pH 7.5), 15 mM
NaCl. The contents were heated at 100oC for 15 minutes and allowed to cool slowly at
room temperature. Dispensed into aliquot and stored at 20oC.
Procedure
5 l/ml RNase solution (the amount of RNase depends upon the RNA contamination)
was added to the crude DNA and incubated at 37oC for 45 minutes.
To the above, 1 ml of TE saturated phenol was added, mixed the contents thoroughly and
then centrifuged at 15,000 x g at 4oC.
The upper aqueous phase was transferred to a fresh tube and added with equal volume of
a mixture of phenol: chloroform: isoamylalcohol (25:24:1) to the solution and mixed
thoroughly without vortexing.
The contents were centrifuged at 15,000 x g at 4oC for 5 minutes and transferred the
upper aqueous phase to a fresh tube.
And to the contents, an equal volume of Chloroform: Isoamylalcohol (24:1) was added
and centrifuge at 15,000 x g at 4oC for minutes. This step is repeated until no precipitate
is seen.
To the 1/10th volume (of the aqueous phase) of 3M Sodium acetate (pH 5.2) was added,
mixed the contents it and then added with twice its volume of chilled absolute ethanol.
Mixed the contents thoroughly by inverting the tubes and incubate at 20oC for one hour
to get precipitate of the DNA.
-
8/4/2019 Protocols of Bio Degradation
22/26
The contents were centrifuged at 15,000 x g for 5 minutes at 4 oC. The DNA was
collected discarding the supernatant and the pellet was air dried to remove ethanol and
dissolved in appropriate volume of TE buffer.
Amplification of the 16s rRNA Gene of the Bacterial Chromosome
The polymerase chain reaction is an enzyme catalyzed biochemical reaction in which
small amount of the specific DNA sequences are amplified into large amounts of linear
double stranded DNA (Millis, 1990). PCR is used to amplify the DNA sequence in
between two known sequences.
The 16s rRNA gene of the bacteria was carried out in our laboratory in the Thermocycler
(Eppendorf). In PCR the specific primers (Forward and reverse primers) complementary
to the known sequences were added along with the master mix (HELINI Biomolecules,
Chennai) to the DNA and the mixture was placed in a thermocycler heated at 94C for
denaturation of the DNA. The mixture is then allowed to cool enabling the primers to
anneal to the complementary sequences. A heat stable DNA polymerase was used to
make the copies of the DNA form the original DNA sequence About 30 cycles of the
DNA amplification was performed that resulted in a very large amplification of the
DNA . The 1542 bp rRNA gene was amplified using two primers. Two primers annealing
at the 5' and 3' end of the 16S rDNA were (Forward Primer) 5'
-GAGTTTGATCCTGGCTCAG-3' (positions 927 [ Escherichia coli 16S rDNA
numbering]) and (Reverse primer) 5'-AGAAA GGAGG TGATC CAGCC-3' (positions
15421525 [E. coli 16S rDNA numbering]) were used. The master mix containing 10X
Taq buffer, 10 mM dNTPs, 25 mM of MgCl2, 1 U ofTaq DNA polymerase,1.5 l of
-
8/4/2019 Protocols of Bio Degradation
23/26
forward primer, 1.5 l of Reverse primer, 100 ng of Genomic DNA and PCR grade
molecular water to make the final volume to 20 l was used. Taq DNA polymerase
initiates the replication of DNA fragments by using nucleotide base from dNTP mixture
(A,T,G,C).
Procedure
3.12.7.1 Setting up the PCR reaction
A fresh master mix tube which contains the dNTP and Taq DNa polymerase was taken
and the following components are added
Master Mix vial 10 l
Forward Primer 1.5 l
Reverse primer 1.5 l
Template DNA 2 l
Nuclease free water 5 l
-------
20 l
-
8/4/2019 Protocols of Bio Degradation
24/26
(Master Mix contains 1U Taq DNA polymerase, 10X Taq buffer, 10 mM dNTPs and
PCR grade water. Primer dye mix contains 10 uM forward primer, 10 uM reverse primer
and PCR compatible dye with glycerol).
After addition of all the components the PCR tube is gently spun down in centrifuge
briefly and is placed in the Thermal cycler. The thermal cycler was programmed as
follows.
Program 1 (one cycle) (Initial denaturation)
94C for 2 minutes
Program 2 (30 cycles) (Amplification)
Step one (denaturation) 94C for 45 seconds
Step two (annealing) 56C for 1 minute
Step three (extension) 72C for 1 minute 30 seconds
Program 3 (one cycle - final extension)
72C for 5 minutes, then hold at 4C
The programmed temperatures of the PCR reaction is represented in the diagram shown
below
-
8/4/2019 Protocols of Bio Degradation
25/26
3.12.7.2 Agarose Gel Electrophoresis
Gel electrophoresis is a technique used for the separation of nucleic acid and protein,
where electrically charged molecules migrate at a rate proportional to their charge in
mass ration when place under an electric field. It is undoubtedly the routine technique to
monitor the success of the nucleic acid isolation procedures and analyze enzymatic
manipulations such as restriction enzyme digestion. About 1.8 g of agarose was weighted
and taken into 100 ml reagent bottle. To it 100 ml of 1X TBE buffer was added and it
was heated in a microwave oven till all agarose gets melted up. The agarose solution was
then poured in to gel casting unit assembled with appropriate comb and it was allowed to
polymerize. After the polymerization the comb was removed and the gel was placed in an
electrophoretic tank consisting of 1X TBE buffer. About 12 l of the 16s rRNA amplified
DNA was mixed with 2 l of the gel loading dye (Bromophenol blue 6x) and it was
loaded in 1.8 % agarose gel The gel was electrophoresed at 90 volts for about 30 minutes
and it was observed in a gel documentation system.
-
8/4/2019 Protocols of Bio Degradation
26/26
3.12.7.3 Eluting DNA from agarose gel fragments
Ethidium bromide stained agarose gel was visualized under a transilluminator on
low setting. The fragment of interest was excised with a clean razor blade. After
removing the excess liquid, the agarose fragment was placed in the spin column.
The tube was centrifuged at 5500 rpm for not more than 45 seconds for the
elution of DNA.
The eluent was checked using a transilluminator for the presence of ethidium
bromide stained DNA.
The eluted DNA was used directly in enzymatic reactions.
This DNA fraction was now subjected for sequencing.
3.12.7.4 Sequencing of Purified Product
The purified product was subjected for DNA sequencing and it was carried out at..
3.12.7.5Sequence match by NCBI Blast analysis
The sequence of the purified product so obtained was subjected to BLAST analysis by using
NCBI database to identify whether the obtained sequence was coding for the related
organisms or not. Confirmation can be brought down by the highest percentage of similarity
with the known sequences in the database.