33

p

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.

34

Map

Fig - 1

35

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

36

Thovala Taluk

12 Tirupatisaram Melur – Perungamadai

13 Tirupatisaram Melur - Temple Pond

14 Tirupatisaram Melur - Chadair

15 Erachakulam

16 Boothapandi

17 Derisanamcope

Table - 2

37

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

38

Vilavancode Taluk

Table - 4

28 Mancode

29 Kuzhithurai

30 Marthandam

31 Methukummal

32 Irayumanthurai

39

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.

40

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.

41

Storage of Samples

Fig - 2

42

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.

43

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.

44

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

45

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.

46

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.

47

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

48

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.

49

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.

50

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.

51

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

52

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.

53

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.

54

· 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.

55

· 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

56

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.

57

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.

58

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

59

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

60

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.

61

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

62

· 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

63

Bioanalyzer

Fig - 4

Bioanalyzer chip

Fig – 5

64

Aligent 2100

Fig – 6

65

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.

66

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.