3. MATERIALS AND METHODS 3.1 COLLECTION SITESshodhganga.inflibnet.ac.in/bitstream/10603/58/6/chapter...
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3. MATERIALS AND METHODS
3.1 COLLECTION SITES
Kanyakumari district, Tamil Nadu, India is at the tip of the Indian
peninsula and faces the Indian Ocean. The district is generally hilly with
plains found near the coast, the land from the sea-coast gradually hilly,
with plains found near the coast. The land from the sea coast gradually
rises from sea level to the Western-Ghats hills in the deep interior of the
district. The district has 62 km of the coast on the western side (Arabian
sea Coast) and 6 km of the coast of eastern side (Gulf of manner/ Bay of
Bengal coast). It is the smallest district in Tamil Nadu with a land spread
of 1,684 sq.km and has almost all ecosystems- forests, wet lands, fresh
water resources, marine, etc. The District, once called the “The Granary
of Travancore” is fertile, with hundreds of water bodies and an excellent
canal irrigation system. Rubber and spice plantations are found on the
hilly terrain, while paddy fields, plantain (banana) and coconut
plantation are found on the plain, near the coast.
The material for study was collected from the different sites in
and around the Kanyakumari district covering rivers, lakes, ponds and
brackish water systems of this district.
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Map
Fig - 1
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COLLECTION SITES
Agasteeswaram Taluk
1 Rajiv Ganthi Nagar - Alamparai
2 Vattatharasi
3 Mandaravilai - Western Side
4 South Vattatharasi
5 Kaniyakullam Paaraiyadi
6 Kaniyakullam Paaraiyadi Pond
7 Kothai Gramam- Thamaarai Kulam
8 Thathair Kulam - near Puthu Gramam
9 Suchindram - Suchindram Aaru
10 Schiedam - near Sri Archanar Temple
11 Manakudi - Kaayal
Table - 1
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Thovala Taluk
12 Tirupatisaram Melur – Perungamadai
13 Tirupatisaram Melur - Temple Pond
14 Tirupatisaram Melur - Chadair
15 Erachakulam
16 Boothapandi
17 Derisanamcope
Table - 2
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Kalkulam Taluk
Table - 3
18 Thiruparappu Aruvi
19 Tiruvattar – Paalaru
20 Aruvikkarai
21 Macode
22 Eraniel - Kaathadi Mooku Valliathankarai
23 Karungal Road - Mathikoodu Pond
24 Trivandrum Main Road - Pond
25 Kaniyakullam Paaraiyadi - Thamaraikullam
26 Vettumadai - Mondaikadu
27 Kalmar – Colachel
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Vilavancode Taluk
Table - 4
28 Mancode
29 Kuzhithurai
30 Marthandam
31 Methukummal
32 Irayumanthurai
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3.2 COLLECTION METHODS
The material for the present study was mainly collected using a
round net (40 cm diameter with a long handle of 350 cm), made of
mosquito net cloth (1mm mesh size). In water bodies with thick
marginal vegetation of aquatic weeds the prawns could be observed
clinging to the weeds. From such ponds, the weeds were dragged out to
the bank and the prawns were hand picked. They could be easily noticed
by their jumping habit as they get dislodged from the weeds. For
collecting the bottom dwelling steel black colored prawns could be hand
picked easily from the spread soil.
The palaemonid prawns of this study could not be collected with
hand net. Instead, a rigorous search under the stones was fruitful for
collecting these prawns. The Palaemonid prawns inhabit the bottom of
the rocks and boulders at the center. These prawns were collected by
turning the stones with care and then hand picked.
For the estimation of population size and relative abundance of
species, collections were carried out throughout the study period, at
regular intervals, strictly between 6.30 to 7.30 am. In each pond certain
definite points were chosen as sites of collection, before the
commencement of study. 10 uniform sweeps were made at each site of
collection. The collections were immediately transferred to polythene
buckets and fixed in 5% formalin. The total number of each counted and
recorded. Very small juveniles were separated under stereoscopic
binocular microscope and counted separately.
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After identifying and confirming the species of genus Caridina
and genus Macrobrachium of Kanyakumari District, special collections
were conducted to decide the availability of the species throughout the
district. With the data collected dominant, less dominant, infrequent
species were decided, according to the number of habitat occupied by
each species.
Adult dissection
For morphometry, the specimens were dissected under the
binocular dissection microscope. At least 200 specimens were examined
for rostra spines, uropod diaeresis and telson spines. Microdissections
using fine needles were performed under binocular microscope on adult
specimens fixed in 5% formalin for morphometric measurements. The
dissected parts were preserved in 5% formalin in separate specimen
bottles for any further reference. A good series of materials of all size
groups were examined to assess the individual variations.
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Storage of Samples
Fig - 2
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Rearing of Adults
Adult animals were reared in mud pots (32 cm diameter; 22 cm
height) and cement tanks (53 cm diameter and 30 cm). These were
observed regularly for few generation and relevant details were
recorded. The temperature in these tanks never exceeded 280C. When
the water level decreased due to evaporation the tanks were filled with
conditioned water. Bottom soil and water weeds brought from the
natural habitat were kept in the respective pots and tanks. Thus, these
artificial tanks were made so meticulously that they almost resembled
the natural habitat. In these tanks where the new species was reared,
pebbles and broken pieces of tiles were kept to provide hiding places for
the shade loving bottom dwelling new species.
Rearing technique of larvae
When hatching took place the larvae were immediately
transferred to separate plastic containers for rearing. The rearing
containers are of 4 cm diameter and 6 cm height. 90% of tap water and
10% of pond water were mixed and kept ready for rearing. The water
was renewed everyday after examining the rearing container for the
presence of moults and dead larvae which were preserved for further
study. No special food was provided as the pond water was added to the
culture medium. The larvae were found to be healthy and active. At each
stage of development, 5 to 10 larvae were preserved for further study.
The larvae were killed by using weak solution of methanol in distilled
water to avoid abdominal flexure and then preserved. The preservative
medium was prepared according to Thakur (1960) which consists of the
following components.
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1. Glycerine -- 5%
2. Formalin -- 5%
3. Distilled water -- 90%
The larvae of each stage were discussed under stereoscopic
binocular microscope. Very fine needles were specially prepared for this
purpose using entomological pins. 5% glycerin solution was used for
dissection. The dissected parts were drawn under a compound
microscope using a cemera-lucida.
Abbreviations used :- r - rostrum, c - Carapace, al - Antennal
Peduncle, ab6 - 6th Abdominal segments, Pr - Propodus, Cp - Carpus, Ch
- Chela, f - Finger, p - Palm and d - Dactylus.
3.3 GENETIC ANALYSIS
3.3.1 ISOLATION OF DNA FROM TISSUE
1. Wash the tissue material twice with the normal saline chop into
fine pieces using surgical blades.
2. Transfer the chopped tissue into 1.5 ml eppendorf tube.
3. Add 500 Micro liters of lysis buffer, 50 Micro liters pf 20% SDS
and 15 Micro liters Proteinase K (conc 20mg /ml).
4. Keep it at 470C for 12-24 hrs to get clear lysate.
5. To the lysate add half of the volume of reagent B. Mix thoroughly
and gently by inverting for 3-4 minutes till the solution becomes
viscous.
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6. Add reagent C (1/4th of reagent B) and mix gently for 3-4
minutes.
7. Add equal volumes (as that of reagents B+C) of phenol +
chloroform (1:1). Mix well and centrifuge at 2500-3000 rpm for
7-8 minutes to separate in to 3 layers viz. aqueous layer, protein
layer and solvent layer.
8. Transfer the aqueous layer carefully into another centrifuge tube
using a broad mouth tip (care should be taken that the protein
layer is not disturbed).
9. Add equal volumes of chloroform to the supernatant and mix
gently for a minute and centrifuge at 2500rpm for 5 minutes.
10. Transfer the aqueous phase to a fresh tube.
11. Add 2 volumes of ice cold Iso Propanol and mix gently to
precipitate the DNA.
12. Spool out the DNA lump in a fresh eppendorf tube and decant
alcohol.
13. Wash the DNA twice with 70% alcohol and give a short spin to
remove alcohol.
14. Dry the pellet properly and ensure that whole alcohol is dried off
15. Dissolve the pellet in 50-100 Micro liters of TE
16. Incubate at 550C for 45 minutes to enhance the dissolution
17. Store the DNA samples at 40C
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Reagent B :
1 M Tris-Hcl (PH-8) - 40ml (40mM)
0.5 M Na-EDTA - 12ml (60mM)
1M NaCl - 15ml (150mM)
Make up to 95 ml with DDW (double distilled water), autoclave then
add 5 ml of 20% SDS(1%)
Reagent C:
5 M Na-Perchlorate (merck)-100gms
Make up to 142ml with DDW (do not autoclave)
TE buffer :
1 M Tris HCl - 1.0 ml (10.0mM)
0.5M EDTA - 0.2 ml( 1 mM)
Make up to 100ml with DDW.
The various chemicals used allow us to separate the DNA from
the cell. First of all, the cell wall needs to break open. The soap /
detergent / extraction solution ruptures the outer part of the cell, while
the salt helps separate DNA from other cellular molecules, such as
carbohydrates. The process of cooling helps protect the DNA from
enzymes that normally do not bother the DNA because the DNA
remains separated from the rest of the cell by the nuclear membrane.
The cold temperatures slow down these enzymes. Filtering removes
most solid matter in the mixture. The last cooling helps the DNA
solidify and precipitate.
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3.3.2 Estimation of DNA concentration and amplification of the
genome
1. The concentration of DNA is estimated using absorbance values
at 280nm in a Nanodrop spectrophotometer.
2. The sample is then diluted to concentration of 25 ng/micro liter
and specific amounts of random primers and DNA Polymerase
enzymes are added.
3. The mixture is then run in a PCR machine for 30-40 cycles and
the amplified 16S rRNA gene product is obtained.
4. The amplified product is then subjected to RAPD and the genetic
difference is observed.
A spectrophotometer is employed to measure the amount of light
that a sample absorbs. The instrument operates by passing a beam of
light through a sample and measuring the intensity of light reaching a
detector.
The beam of light consists of a stream of photons, represented by
the purple balls in the simulation shown below. When a photon
encounters an analyte molecule (the analyte is the molecule being
studied ), there is a chance the analyte will absorb the photon. This
absorption reduces the number of photons in the beam of light, thereby
reducing the intensity of the light beam.
Nanodrop Spectrophotometer
The NanoDrop is a cuvette free spectrophotometer. It uses just 1
micro liter to measure from 5ng/ml. The Nanodrop has greatly improved
our sequencing success rates and our ability to troubleshoot problems
more effectively.
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Nanodrop Spectrophotometer
Fig. - 3
Key features
1ml sample and no cuvette
Large dynamic range 5 ng/ml to 3,000 ng/ml
Quantitation of DNA, RNA and proteins
Low volume spectrophotometer
Instrument Description
The NanoDrop ND-1000 is a full-spectrum (220-750nm)
spectrophotometer that measures 1 ml samples with high accuracy and
reproducibility. It utilizes a patented sample retention technology that
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employs surface tension alone to hold the sample in place. This
eliminates the need for cumbersome cuvettes and other sample
containment devices and allows for clean up in seconds. In addition, the
ND-1000 has the capability to measure highly concentrated samples
without dilution (50X higher concentration than the samples measured
by a standard cuvette spectrophotometer).
Operation
The sample retention system used by the NanoDrop ND-1000 is
one of the key features of the instrument. Not only does it enable the
analysis of 1ml samples, it also eliminates the need for cuvettes and
capillaries. This saves on the cost of disposable cuvettes and/or on the
time and effort spent in cleaning reusable ones. For busy laboratories,
this feature alone can pay for the cost of the ND-1000.
With the sample apparatus open, a droplet of sample is pipetted
onto the measurement pedestal.
When the sample apparatus is closed, the sample arm slightly
compresses the droplet and a sample column is drawn. Surface tension
alone holds the sample in place. The spectral measurement is then made
and quantification is made based on the tightly controlled path length of
1mm.
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When the measurement is complete, the sample apparatus is
opened and the sample is simply wiped from both the sample arm and
sample pedestal using an ordinary dry laboratory wipe.
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Application
UV/VIS spectrophotometer is simple for samples as small as 1ml
using the NanoDrop ND-1000 Spectrophotometer. The small sample
requirement and ease of use, make the NanoDrop ND-1000
Spectrophotometer ideally suited for measuring:
Nucleic acid concentration and purity of nucleic acid samples up
to 3700 ng/ml (ds DNA) without dilution
Fluorescent dye labeling density of nucleic acid micro array samples
Purified protein analysis (A280) up to 100 mg/ml (BSA)
Expanded spectrum measurement and quantitation of fluorescent
dye labeled proteins, conjugates and metalloproteinase
Bradford Assay analysis of protein
BCA Assay analysis of protein
Lowry Assay analysis of protein
Cell density measurements
General UV-Vis spectrophotometry
3.3.3 Amplification of DNA
The all employ a widely used molecular technique called
polymerase chain reaction or PCR.
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The idea was conceived by Kary Mullis in the early 1980s and
was first described, albeit briefly, in an article investigating the mutation
that causes sickle cell anemia [1]. The details of the method and its uses
were discussed in greater detail over the next two years [2-3]. PCR
revolutionized molecular genetics by allowing rapid duplication and
analysis of DNA.
The PCR method
PCR is used to amplify a specific region of DNA in order to
produce a large number of nearly identical copies. The method uses a
heat stable DNA replication enzyme called a DNA polymerase, the four
deoxynucleotide building blocks of DNA and two small single-stranded
DNA segments called primers, which flank the “target” region of DNA
to be amplified and are complementary to each strand (meaning the
matching strand to which its bases pair).
There are 3 basic steps in PCR that are carried out at different
temperatures to create conditions optimal for:
DNA denaturation (meaning to separate the double-stranded DNA
into single strands).
Primer binding or hybridization to each of the single strands of
DNA at either the beginning or the end of the target sequence,
depending upon the single-strand of DNA. Hybridization
combines complementary, single-stranded DNA into a single
molecule. This process is called annealing.
DNA polymerase elongation. The enzyme attaches to the
primersingle-stranded
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DNA duplex and synthesizes the complementary strand of DNA,
using the existing single-strand as a template.
Newly synthesized DNA strands can serve as additional template
for complementary strand synthesis. PCR rapidly amplifies DNA;
because both strands are copied, there is an exponential increase in the
number of copies. Assuming there is only a single copy of the target
gene before cycling starts:
Cycle Single-strand Copy Number
Cycle 1 4 copies (22)
Cycle 2 8 copies (23)
Cycle 3 16 copies (24)
… …
Cycle 35 68.7 billion copies (236)
After 35 cycles of PCR, there will be over 68 billion copies! In
reality, PCR starts with many copies of the target gene, so the end result
is typically higher. Each cycle only takes a few minutes. Factoring in the
time to change temperatures, the entire process can be done in several
hours.
RAPD stands for Random Amplification of Polymorphic DNA. It
is a type of PCR reaction, but the segments of DNA that are amplified
are random. The scientist performing RAPD creates several arbitrary,
short primers (8-12 nucleotides ), then proceeds with the PCR using a
large template of genomic DNA, hoping that fragments will amplify. By
resolving the resulting patterns, a semi-unique profile can be gleaned
from a RAPD reaction.
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No knowledge of the DNA sequence for the targeted gene is
required, as the primers will bind somewhere in the sequence, but it is
not certain exactly where. This makes the method popular for comparing
the DNA of biological systems that have not had the attention of the
scientific community, or in a system in which relatively few DNA
sequences are compared. Because it relies on a large, intact DNA
template sequence, it has some limitations in the use of degraded DNA
samples. Its resolving power is much lower than targeted, species
specific DNA comparison methods, such as short tandem repeats. In
recent years, RAPD has been used to characterize, and trace, the
phylogeny of diverse plant and animal species.
Random Primers
1 5’-GGT-GCG-GGA-A-3’
2 5’-GTT -TCG-CTC-G -3’
3 5’ -GTA-GAC-CCG-T-3’
4 5’ -CAG-AGC-CCG-T-3’
5 5’ -AAC-GCG-CAA-C-3’
6 5’ -ACC-GTC-AGC-A-3’
Table - 5
Multiplex PCR
Multiplex PCR is a powerful technique enabling amplification of
more target genes (pathogens) in a single reaction.
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· All the pathogens will be diagnosed by Polymerized chain
reaction based techniques and the results will be projected
through electropherogram and gel like image by special scanner
and it has been compared and verified by computer software.
· High resolution of a large number of bands which is critical for
any multiplex PCR application ie., comparative gene expression
analysis, pathogen detection etc.,
· Broad linear dynamic range : enables the detection of less
abundant products, e.g., low abundance messages in multiplex
RT-PCR amplification or non-specific amplification. Excellent
quantitation of PCR products over a large linear dynamic range is
thus achieved.
· High sensitivity and specificity : higher sensitivity and specificity,
broad linear dynamic range and sharpness of bands enables the
detection of less abundant products that are not visible in an
agarose gel (e.g. non-specific amplifications or carry-over
products).
· Sensitivity : LIF detection for fragments down to 0.1 ng/ml.
Minimal sample consumption and fast results.
· Quick and easy sample comparison. One click overlay, scaling or
zooming features.
· Improved sizing accuracy : Normalization to two internal markers
and a ladder provides accurate and reproducible sizing.
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· Automated and reproducible quantitation. The special software
quantitates each DNA fragment automatically against internal
standards. This ensures increased accuracy and reproducibility.
· Various data display options. Digital data for convenient archiving
and storage for future reference.
Product specifications
Enzyme:
QIAGEN Taq DNA Polymerase is a recombinant 94 kDa DNA
polymerase. Deoxynucleoside-troposphere: DNA deoxynucleotidyl
transferase, EC 2.7.7.7, originally isolated from Thermus aquaticus, and
expressed in E.coli.
One unit of Taq DNA polymerase is defined as the amount of
enzyme that will incorporate 10 nmol of dNTPs into acid-insoluable
material within 30 min at 720C, under the assay conditions described in
the section quality control on the following page.
Concentration: 5 units/ml
Substrate analogs: dNTP, ddNTP, dUTP, biotin-11-dUTP, DIG-11-dUTP,
Fluorescent dNTP/ddNTP
Extension rate : 2-4 kb/min at 720 C
Half-life: 10 min at 970 C; 60 min at 940 C
5’-3’ exonuclease activity : Yes
Extra A addition : Yes
3’-5’ exonuclease activity : No
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Nuclease contamination : No
Protease contamination : No
RNase contamination : No
Self-priming activity : No
Storage and dilution buffer: 20mM Tris HCl, 100mM KCl, 1mM
DTT, 0.1 mM EDTA, 0.5% (v/v) Nonidet P-40, 0.5% (v/v)Tween 20,
50% (v/v) glycerol; pH8.0 (200C)
Buffers and Reagents:
QIAGEN PCR Buffer: 10X concentrated MgCl2 : pH 8.7 (200C)
Q-Solution; 5x concentration MgCl2 solution : 25mM
dNTP Mix : 10mM each of dATP, dCTP, dGTP and dTTP; ultra pure
quality.
Distilled water : Ultra pure quality; PCR-grade
Taq PCR Master Mix:
Taq PCR Master Mix: 2X concentrated. Contains Taq DNA Polymerase,
QIAGEN PCR Buffer (with 3mM MgCl2) and 400mM of each dNTP
PCR Protocol Using Taq DNA Polymerase
This protocol serves as a guideline for PCR amplification.
Optimal reaction conditions, such as incubation times, temperatures and
amount of template DNA, may vary and must be individually
determined.
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Important notes before starting
Set up all reaction mixtures in an area separate from that used for DNA
preparation or PCR product analysis.
Use disposable tips containing hydrophobic filters to minimize cross-
contamination.
If required, prepare a dNTP mix containing 10mM of each dNTP. Store
this mix in aliquots at -200C. For convenience, the Taq PCR Core Kit
includes a ready-to-use ultra pure dNTP Mix, and Taq DNA
Polymerase, QIAGEN PCR Buffer and dNTPs.
Protocol
1. Thaw 10X QIAGEN PCR Buffer, dNTP mix, primer solutions
and 25mM MgCl2 (if required)
Keep the solutions on ice after complete thawing. Mix well before use to
avoid localized differences in salt concentration.
2. Prepare a master mix according to Table 6
The master mix typically contains all of the components needed for PCR
except the template DNA. Prepare a volume of master mix 10% greater
than that required for the total number of PCR assays to be performed. A
negative control (without template DNA) should be included in every
experiment. The optimal Mg2+ concentration should be determined
empirically but in most cases a concentration of 1.5mM, as provided in
the 1x QIAGEN PCR Buffer, will produce satisfactory results. Keep the
master mix on ice.
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Table 6. Reaction composition using Taq DNA Polymerase
Component Volume/reaction final concentration
Master mix
10xQIAGEN PCR Buffer 10ml 1x
25mM MgCl2 Variable see table 2
dNTP mix (10mM each) 2ml 200mM of each dNTP
Primer A Variable 0.1-0.5 mM
Primer B Variable 0.1-0.5 mM
Taq DNA Polymerase 0.5ml 2.5 units/reaction
Distilled water
Template DNA
Template DNA, added at step 4 variable <1 mg/reaction
Total volume 100 ml
Contains 15mM MgCl2
Table 7. Final Mg2+ concentrations
Final Mg2+ concentration in reaction (mM): 1.5, 2.0, 2.5, 3.0, 3.5, 4.0,
4.5, 5.0
Required volume of 25mM MgCl2 per reaction (ml): 0,2,4,6,8,10,12,14
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3. Mix the master mix thoroughly, and dispense appropriate
volumes into PCR tubes.
Mix gently, for example, by pipetting the master mix up and down
a few times. It is recommended that PCR tubes are kept on ice before
placing in thermal cycler.
4. Add template DNA (<1 mg/reaction) to the individual tubes
containing the master mix.
For RT-PCR, add an aliquot from the reverse transcriptase
reaction. The volume added should not exceed 10% of the final PCR
volume.
5. When using a thermal cycler with a heated lid, do not use
mineral oil. Proceed directly to step 6. Otherwise, overlay with
approximately 100 ml mineral oil.
6. Program the thermal cycler according to the manufacturer’s
instructions.
A typical PCR cycling program is outlined in Table 3. For
maximum yield and specificity, temperatures and cycling times should
be optimized for each new target or primer pair.
Table 8. Thermal cycler conditions
Additional comments
3-step cycling
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PCR Cycling Details :
Denaturation : Denaturation was done at a temperature at 950C for
a period of 3 minutes
Annealing : before the annealing step PCR was run at 940C for 30
seconds and was run at 350C for 90 seconds
Elongation : elongation was carried out at 720C for 90 seconds.
The following steps were repeated for 40 cycles and finally the
samples were run at 720C for 10 minutes.
For a simplified hot start, proceed as described in step 7.
Otherwise, place the PCR tubes in the thermal cycler and start the
cycling program.
7. Simplified hot start : Start the PCR program. Once the thermal
cycler has reached 940C, place the PCR tubes in the thermal cycler.
In many cases, this simplified hot start improves the specificity of
the PCR. For information on highly specific and convenient hot-start
PCR using HotStarTaqTM DNA Polymerase.
Note: After amplification, samples can be stored overnight at 2-80C, or
at -200C for longer storage.
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3.4 ELECTROPHORESIS
Principle
Electrophoresis is a technique used to separate and sometimes
purify macromolecules - especially proteins and nucleic acids - that
differ in size, charge or conformation. As such, it is one of the most
widelyused techniques in biochemistry and molecular biology.
When charged molecules are placed in an electric field, they
migrate toward either the positive or negative pole according to their
charge. In contrast to proteins, which can have either a net positive or
net negative charge, nucleic acids have a consistent negative charge
imparted by their phosphate backbone, and migrate toward the anode.
Proteins and nucleic acids are electrophoresed within a matrix or
"gel". Most commonly, the gel is cast in the shape of a thin slab, with
wells for loading the sample. The gel is immersed within an
electrophoresis buffer that provides ions to carry a current and some
type of buffer to maintain the pH at a relatively constant value.
DNA 1000 Lab Chip Kit
· 25 DNA chips
· 1 Electrode Cleaner
· DNA 1000 Ladder
· DNA 1000 Markers 15/1500 bp
· DNA dye concentrate
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· DNA gel matrix (3 vials )
· 3 Spin filters
· 1 Syringe
Bioanalyzer
The Agilent 2100 bioanalyzer is a personal Lab-on-a-chip
platform operating disposable micro fluidic chips for analysis of
DNA/RNA/Proteins and Cells. For the last 4 years, the Agilent 2100
bioanalyzer has replaced gel electrophoresis for the analysis of DNA
fragments in many laboratories around the world. It is currently used for
GMO detection, target validation via end-point PCR, pathogen
detection, mutation analysis, and micro satellite analysis .
The bioanalyzer is an ultra sensitive and flexible CCD camera
based instrument for imaging micro arrays and related formats. It is
suitable for fluorescence as well as chemiluminescence imaging.
Advantages:
· Ultra stable white light source: Free choice of excitationwavelength by motorized filter wheel
· No restriction in the choice of the dye
· User programmable software for acquisition, analysis anddocumentation
· Segmentation algorithm and feature valuation gives a set offeature quality parameters
· Dust recognition and elimination
· Fast data acquisition
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Bioanalyzer
Fig - 4
Bioanalyzer chip
Fig – 5
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Aligent 2100
Fig – 6
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Method:
Preparing the Gel-Dye Mix:
1. Allow DNA dye concentrate (blue �) to a DNA gel matrix (red �)
to equilibrate to room temperature for 30 min.
2. Add 25 ml of DNA dye concentrate (blue �) to a DNA gel matrix
vial (red �).
3. Vortex solution well. Transfer to spin filter.
4. Centrifuge at 2240g + 20% for 15 min. Protect solution from
light. Store at 40C. Use within 4 weeks.
Loading the Gel-Dye Mix
1. Allow the gel-dye mix equilibrate to room temperature for 30 min
before use.
2. Put a new DNA chip on the chip priming station
3. Pipette 9.0 ml of gel-dye mix in the well marked.
4. Close the chip priming station
5. Press plunger until it is held by the clip.
6. Wait for exactly 60s then release clip.
7. Wait for 5s. Slowly pull back plunger to 1 ml position.
8. Open the chip priming station and pipette 9.0 ml of gel-dye mix in
the wells marked.
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9. Pipette 5.0ml of the gel-dye mix in the well marked with the
ladder symbol.
Loading the Markers
1. Pipette 5 ml of marker (green �) in all 12 sample wells. Do not
leave any wells empty.
Loading the ladder and the samples
1. Pipette 1ml of DNA ladder (Yellow �) in the well marked
2. Pipette 1 ml of sample in each of the 12 sample wells.
3. Put the chip in the adapter and vortex for 1 min at the indicated
setting (2400 rpm).
4. Run the chip in the Aglient 2100 bioanalyzer within 5 min.