EXPERIMENT 20

plant population density by quadrat

Tostudy method.
REQUIREMENTS
Thread, nails, hammer, metre scale, paper,
pencil.
THEORY
Populationis defined as total number of individuals
of aspecies
similar resources and can interbreed. Population ecology found in a given
is an important area ofgeographical area; sharing
ecology because it links
ecology to population genetics and evolution. Size of population may be outcome of competition with another
species,impact of predator or effect of use of pesticides. Size of population keeps changing, depending upon -
(a) Food availability (b) Predation (c) Weather
A Natality: Numbers of births during a given period in the population. It
increases population density.
la) Mortality: Number of deaths in a population during a
given period.
Immigration: Number of individuals of same spp. coming into the habitat from elsewhere.
(o) Emigration: Number of individuals of population who left the habitat and moved to other habitats.
So population density at a time "t" is given by:
N, = N, + [(B + I) - (D+ E)
N = Population density at time o.
N, = Population density after time t.
B= Birth rate
= Immigration rate.
D = Death rate.
E = Emigration rate.
No. of individuals (N) in an area
Population density =
Area (S)

N
D= - No. of individuals of a species present per unit area.

Population density can be studied by laying quadrats of size 1 sq. m. and recording number of individuals
of different species in the given quadrat.
Quadrat: Sampling plot or area generally of size 1 m x1m. Chosen randomly, as representative of the given
area and species falling inside the quadrat are recorded to find out population density.
Population ecology: It is the study of individuals of same species including processes like interdependence
and factors governing it.

PROCEDURE
L. Select a quadrat randomly in a uniform area.
a. With the help of metre scale, make a quadrat of lm x Im in the field. Fix 4 nails at the corners of the
quadrat and tie/fix thread or string over the nails.
O Count the number of plants of each species present in the quadrat. If the plant is of small size and large
n number, then each quadrat can be further divided into smaller units.
Experiments 67
 of plants in that quadrat also.
4. Make another quadrat in the same area and record number
5. Repeat with the third quadrat.
1m Nail

Y
String
Species A

-Species B

33
Y Species C

1m Y-Species D
1m

-Species E

O8-Species F

1m

A
quadrat with subunits

OBSERVATIONS

No of individuals in the area Im x 1 Total number of Average

S.No. Name of plant spp. individuals in 3
m quadrats
quadrats
III

A
B
C
D

RESULT
in an area.
Population density of a plant spp. is the average no. of individual plants of the spp. occurring
Total no. of individuals in 3 squares
Population density =

PRECAUTIONS
1. Count the individuals of one plant spp. at a time.
2. Field chosen should have uniform distribution of spp. towards inside.
3. Plant lying under the string should be considered in quadrat, if more than half of it lies

68 ogether wek Biology Lab Manual-XIl

 EXPERIMENT 21
ALM
plant population frequency by quadrat method.
study
To
BQUIREMENTS
Thread/string, nails, hammer, metre scale, paper, pencil.

THEORY
area. Number
Populationis defined as total number of individuals of a species found in a given geographical like birth
individuals in a population never remains constant. It may increase or decrease due to many factors
of migration, etc.
rate,death rate, species occurs and
Population frequency refers to number of sampling units (quadrats) in which a given
expressesthe distribution or dispersion of various species in a community.
thus
Number of quadrats of occurrence of spp. x 100
Population Frequency =
Total no. of quadrats studied
studied because in smaller units. individuals of different
The guadrat of size Im x Im can be divided into smaller units and
more accuratelv.
species can be counted
PROCEDURE
1Select asite and with the help of metre scale lay a quadrat.
nails.
2. Tie string on the tying strings at distance of 25 cm each on either
3 Each quadrat can be divided into 16 small squares by
side Smaller squares can also be marked with nails and strings.
4 Note down the number of plants of each spp. in each square.
5 Find the total number of plants of each spp. in each quadrat.
6. Select another quadrat randomly and repeat the steps.
OBSERVATIONS
No. of individuals of spp. present in Total no. of Frequency %
S.No. Name of plant spp. quadrats in which
each quadrat
spp. occurred A)
=4Ax 100

III IV

ASULT
Number of quadrats in which species appeared x 100
The frequency percentage for a spp. = Total number of quadrats studied

The plant species, has highest frequency so it is the dominant species.

Frequency of a species gives information about:
0 1ype of habitat and correlation of plant with habitat.
(2) Interaction of spp. with other species.
Dudy of quadrats are taken as representatives of the complete area.
Experiments 69
RECAUTIONS
1. Measure the quadrat accurately.
2. Quadrats should be studied from 1 area only, with uniform distribution of plants.
3. If a plant has more than half of its portion inside the quadrat; it should be assumed and
the quadrat.
1m

1m 1m

1m

(1m x 1m) Quadrat with subunits

VIVA VOCE
Experiments 20 and 21
Q.1. Define ecology. Q.4. Why do we study quadrats?
Ans. It is the branch of science which deals with Ans. Quadrats are taken to be representative o e
the study of relationships between the living whole area in which they are studied and
organisms and the physical factors of the give an idea of the type of vegetation in agi
environment surrounding them. type of soil and habitat.
Q.2. What are the physical factors of environment?
Q.5. Can we take a quadrat of shapes like ar
Ans. Air, water, land, temperature, humidity, type rectangle, etc.?
of soil and climate.
Ans. Yes. We can take a circle or rectangle ¿
Q.3. What is the difference between community and sampling unit because we want an area i
population? represent vegetation.
Ans. Population is a group of individuals belonging to Q.6. What is vegetation?
same species in a given area whereas community
Ans. Plant growth in totality including small or lar*
involves number of spp. living together in a certain
environment and interacting with each other. population of each spp.

70 7ogether witk Biology Lab Manual-XlI

 EXPERIMENT 24
AIM

To study the different stages of mitosis in onion root tip and grasshopper larva from temporary o
permanent slides.

REQUIREMENTS
Onion root tips, larva of grasshopper, slides, coverslips, filter papers, acetocarmine stain, dissecting needles
forceps, water, watch glass, microscope,spirit lamp, incubator, 1 NHCI, acetic acid, methanol, formalin.
THEORY
German Biologist Rudolf Virchow, first conceived the idea that cells arise from pre-existing cells only. Growth
requires an increase in cell mass, a duplication of genetic material and a division assuring that each daughter
cell receives an equal complement of the genetic material to ensure perpetuation of the cell line. Thus doubling
and halving genetic material, cycle of cell growth is known as cell cycle. In life of a cell, capable of division, cell
division is but a phase of shortest duration; rest is the phase in which cell grows and synthesizes genetic and
other material that are passed on to daughter cells.
Cell cycle consists of four stages, i.e., Gi, S, G2, and M or D.
G1 or Gap Phase I: Period of growth and increase in cell mass, chemical preparation for DNA synthesis.
It is the largest phase. It lasts for 6-8 hours.
Sor Synthetic phase: The cell synthesizes genetie material-DNA from raw materials and hence DNA gets
duplicated. It lasts for 5-6 hours.
G2 or Gap phase II: Cell duplicates materials other than DNA to be passed on to daughter cells like
mitochondria, Golgi apparatus, cytoplasmic inclusions. It lasts for 4-5 hours. G,, S and G, phases together
constitute interphase.
M phase or D phase: The cell divides during this phase and it lasts for 1hour. M phase may consist of
mitosis or meiosis.
Cell division has two parts:
1. Karyokinesis: The division of nucleus into two nuclei is known as karyokinesis.
2. Cytokinesis: The division of cytoplasm into two daughter cells is known as cytokinesis.
Karyokinesis is of two types: (i) Mitosis (Equational division) and (ii) Meiosis (Reductional
Mitosis: Mitotic cell cycle in animals was first described by W. Fleming in 1882 division).
described by Strasburger, again in 1882. Mitosis is a kind of cell division where two and in plants, it was rs.
which are genetically identical to each other as well as to the daughter cells are produced
of unicellular as well as multicellular parents cell. It is thus, the basis of continuatio
of worn out tissues. organisms. It occurs in somatic cells, helping in growth as well as repd1
Meiosis: Meiosis takes place in the germ
cells are produced which have half the numbercells to produce gametes. As a result of
of chromosomes as the parent cell and meiosis itfour daugne
reductional division. hence is known

PROCEDURE
I. To Make Slide of Onion Root Tips:
1. Remnove the old roots from onion and
put it in bottle full of water. New roots
2. Cut the root tips when the roots are appear in 4-5 ay
about 2-3 cm in length.
3. Root tips are then transferred to FAA ixative for 24 hrs. [FAA fixative = 40 %formalin: 90%
alcohol: Glacial acetic acid in the ratio of1:9: 5]
T
 II. To Make Slide of Grasshopper Larva:
1. Put the grasshopper larva in a vial containing mixture of acetic acid: methanol in
24 hours for fixation.
the ratiy f1
2. Take out a piece of larval tissue on aslide. Add acetocarmine to it and warn the slide
lamp.
3. When the tissue is sufficiently stained, place coverslip over it.
4. Gently tap the tissue with the help of a needle or thumb for uniform distribution of cel.
5. Observe it under low power and then under high power of microscope.

OBSERVATIONS
Mitosis is defined as A process in which the chromosome of a nucleus split longitudinally
and their split halves migrate to the two poles constituting two daughter nuclei which are into
other as well as to the parent nucleus both qualitatively and quantitatively." identical
The process of mitosis is completed under the following stages:
1. Interphase
() Stage in between two successive cell divisions and hence a non-dividing phase.
(ü) Nucleus large and distinct with nuclear membrane.
iü) Nucleolus clearly visible.
(iw) Nucleus contains chromatin network.
2. Prophase
() Nucleus enlarged and occupies most of cell volume.
(ü) Chromatin network gets condensed and appear as long thread-like structures called
chromosomes
(ii) Each chromosome consists of two chromatids held together by centromere.
(v) Nucleolus gradually disappears by the end of prophase.
(vij) Nuclear membrane starts disappearing.
In animal cells, the centrioles move to the opposite poles and the
centrosome forms aster ravs.
8. Metaphase
(i) Nuclear membrane completely disappear.
(ü) Chromosomes become shorter and thicker and hence
become distinct and clearly visible under :
compound microscope.
(iüi) Chromosomes orient themselves towards the
equator with their centromeres arranged on a equatora
line forming metaphase plate. Out of two chromatids,
faces the opposite pole. one faces one pole and the other chromaa
(iv) Series of spindle fibres attach the
In animal cells, the spindle fibres
centromeres to the opposite poles.
in the general cytoplasm.
originate from centrioles whereas in plant cells, they are synthesllr.
4. Anaphase
() Centromere of each chromosome divides into two so that each
(i) Spindle fibres gradually shorten so that chromatid gets its own centrous
each chromatid with its towards is
centromere isarmspulled
respective pole with the centromere proceeding towards poles and chromosome trailing behund
(iü) Each chromatid now behaves
as an independent
chromOsome. chromosome and is thus known as daughter
5. Telophase
) At each pole, the
Thus chromosomesdaughter
are again chromosomesstart uncoiling, elongate and thin and
invsible
(ii) Nuclear reorganized into chromatin network. become
membrane reappearS.
(iü) Nucleolus gets
reconstituted.
84 Together wtk Biology Lab
Manual-XII
 of onion should be in nally th
1. Base
2.
Filter the acetocarmine contact with water
stain
Material should be before use. during the growth of
the slide and warmed gently. Do not
3. roots.
4. Clean coverslip allow the solution to
and tclean.
horoughl y
should be neat boil.
5. The slide
before and after use.
6. Material should be mounted in the
7. There
should be no air centre of slide.
bubble under the
coverslip.
Nuclear membrane
Chromatin fibres
-Nucleolus
-Cell membrane

-Cell wall

Interphase
Cell wal!
-Nuclear membrane
Nuclear membrane
-Nucleolus -Disappearing nucleolus
Chromosomes -Chromosomes

-Cell wall
Early Prophase Late Prophase

-Spindle fibres
Daughter chromosome
Chromosomes with
Chromatids
Spindle fibres

Early Anaphase
Metaphase
-Daughter cells
Cell wall Daughter nuclei
Daughter chromosomes Cell plate
(Chromatids) Nucleolus
Nuclear membrane
Spindle fibres

Telephase and Cytokinesis

Late Anaphase
onion root tip cells (Plant cell)
Fig. Stages of mitosis in

Expe=
 EXPERIMENT 25
AM
testis
To study the various stages of meiosis in onion lower buds or grasshopper
permanent slides. throngh
REQUIREMENTS
Permanent slides showing different stages of meiosis; microscope.

THEORY
Meiosis is atype of cell division where the chromosome number is reduced to half and thus it is also kn
as reductional division. As a result of meiosis, 4 cells are produced with half the number of chromosomes
in parent cell.
The important events during meiosis are:
Synapsis, i.e., pairing of homologous chromosomes.
" Crossing over, ie., exchange of genetic material bet ween non-sister chromatids of homologos
chromosomes.
" Reduction of chromosome number to half.
Significance of Meiosis :
1. In sexually reproducing organisms, gametes fuse to form zygote and these gametes are formed by me.s
to maintain constant number of chromosomes in each generation as meiosis reduces the chromosome
number tohalf in gametes and fertilization, i.e., fusion of gametes restores the diploid or paired condi
of chromosomes.

2. Crossing over leads to new combinations of genes or variation thus helping in evolution.
PROCEDURE

L For Temporary Mount of Grasshopper Testis:

1. With the help of forcep, take out testis lobe on a clean slide in a drop of
acetocarmine.
2. Warm the slide over spirit lamp for a minute.
3. After 4 - 5 minutes wash off extra stain with blotting paper and
add a drop ofglvcerine.
4. Place a coverslip over it and press it gently with a thumb after keeping the slide
blotting paper to separate the cells. between folds
5. Observe the slide under microscope.
II. For Temporary Mount of Onion Flower Buds:
1. Onion buds are placed in fixative which contains
acetic acid and methanol in the ratio 3: 1
2. Open up afloral bud and extract the anthers.
3. Tease these anthers on a slide containing few
drops of acetocarmine.
4. Warm the slide over spirit lamp for a minute.
5. Put the coverslip and squash it by pressing it with thumb or back of a
slide between folds of filter paper. dissecting needle after keep
6. Observe the slide under microscope.
.
Zygotene Pachytene

Metaphase I
Diakinesis
OBSERVATIONS
of two divisions Meiosis I and Meiosis II. Meiosis Iis the reductional part of the division
Meiosis consists
thechromosome number is reduced to half. Meiosis II is the eguational division similar to mitosis.
as
Meiosis I
Stages of
Meiosis I is divided into the following stages:
1. Prophase I
. It is divided into five substages.
Leptotene
() Chromosomes appear as long thread like structures.
(ii) Chromosomes show beaded appearance due to chromomere.
(iii) Nuclear membrane is clearly visible.
Zygotene
() Chromosomes are short and thick.
bivalent. Pairing of homologous chromoSomes
(ii) Homologous chromosomes start pairing and form a
is known as synapsis.
Pachytene
form bivalents or tetrads.
(i) Pairing of homologous chromosomes is completed and they chromosomes.
chromatids of homologous
(i) Crossing over takes place between non-sister
Diplotene
(i) Paired chromosomes start separating.
point of crossovers.
(i)) Chromosomes are held together at chiasmata, i.e., the
Plasma membrane
Centrioles

Centrioles
Plasma membrane

Nucleolus
Nuclear mernbrane

Chromosomes Homologous
chromosomes

Nucleolus Nuclear
membrane

Leptotene Zygotene

Tetrad
Centrioles Centrioles
Nuclear
membrane Degenerating
nuclear membrane
Nucleolus
Homologous Crossed over
chromOSomes
homoBuyous
showing chromOsomes
synapsiS
Nucleolus

Pachytene Diplotene

Experiments 91
 EXPERIMENT 27
AIM
To study
pedigree charts of genetic traits like rolling of tongue, blood groups, ear lobes, widow's
peak, colour blindness.

REQUIREMENTS
Pedigree charts for various traits.
THEORY
Pedigree: A record of inheritance of certain genetic traits for two or more generations presented in
form of a diagram or family tree is called pedigree. It is employed in human beings and domesticated animal
mainly pets. Because (a) controlled crosses/mating cannot be carried out in human beings (b) generation ti.
is long (c) number of offsprings per couple is small. So the inheritance of traits in humans can be studied h.
pedigree analysis.
Advantages of Pedigree Analysis
() Pedigree analysis indicates Mendel's principles are also applicable to human genetics with soma
modification.
(ii) It helps tofind whether the trait is dominant or recessive.
(ii) It helps the genetic counsellors to advice the intending couples about the possibility of having children
with genetic defects like haemophilia, colour blindness, sickle cell anaemia, etc.
(iv) Carriers cannot be identified unless an affected child is borne to the carrier individual or without studying
the pedigree.
(v) It helps in predictions regarding a character in children if related individuals or those with a famil;
history for such traits are married.
(vi)) Recessive traits are expressed in cases where both the parents are closely related.
Symbols Used in Pedigree Analysis
Symbol Meaning of Symbol
Circle Female individual

Square Male individual

Horizontal line connecting circle and square Marriage
Line perpendicular to marriage bar Offsprings

or
Shaded square or circle Affected individual
Half shaded square or circle
Heterozygous foracharacter
or Dot in middle of square or character
circle Carrier of sex linked recessive

Rhombus with a number unspecified

Offsprings where sex is
and no. indicates no, of offspr1n8
Two horizontal lines relatives.
connecting square and circle Marriage between close
 CommonGeneticTraits
Taster:PTC (Ge., Phenyl thiocarbamide) paper has abitter taste. It is a genetic dominant trait.
PTC
1.Genotype
TT.and Tt are tasters of PTC whereas tt are non-tasters, ie, they do not taste PTC paper as hbitter.
Rolling of Tongue: It is the ability of an individual to rollthe tongue into U-shaped tube. It is also a
dominant trait and follows Mendelion laws of inheritance. Non-rollers are homozygous recessive.

Tongue roller Non-roller

Tongue Rolling Trait
[' are
Blood Groups: It is a case of multiple alleles as there are 3 alleles-IA, JB, and [º. IA and
co-dominant whereas IA and I are completely dominant over [º.
widow's
Widow's Peak: Ahair ine that forms distinct peak as it crosses the forehead is said to have
have V-shaped front hair line whereas
peak. The homozygous dominant or heterozygous individuals
homozygous recessive individuals have straight hair lines.

Straight hairline
Widow's peak
individual as the
Linked Inheritance: The inheritance of such traits depend on the sex of the
5. Sex for
chromosome X. Y chromosome does not carry any allele one
alleles for these traits are present on sex compared to females because males have only
these traits. Such traits are more visible in males as
haemophilia.
allele for that trait. eg, Colour blindness,
to recessive gene.
6. Fused Ear Lobe: It is a trait due
categorized as follows:
The pedigree analysis can be
Pedigree seldom skips a generation.
(i) Autosomal dominant trait ’
Pedigree may skip a generation.
(ü) Autosomal recessive trait ’ common in females.
trait More
(iii) Sex linked dominant males.
trait More common in
(iv) Sex linked recessive

PRECAUTIONS
traits.
pedigree chart of various
1. Observe the individuals,
the genotypes of the
2. Try tofind out blindness
Chart for Colour
I. Pedigree

Experiments 101
 EXPERIMENT 29
organs in various plants and animals.
study homologous
To
REQUIREMENT
and models.
Narious specimens

THEORY
functions.
are the organs which have common origin and similar basic structure but perform different
Homologous organs
Plants
Homologous Organs in
plant and spines of barberry:
Tendril of pea
These have cocommon origin as they arise from axillary position but they are
homologous organs as perform
meant for protection.
functions. Tendrils provide mechanical support to a plant whereas spines are
diferent

Tendril

Spine

Leaf spine in barberry

Leaf tendril of pea

Fig. Homologous organs in plants

Homologous Organs in Animals

of man:
Forelimbs of horse, wings of bat, flippers of seal and hands
of man are examples of homologous
Forelimbs of horse, wings of bat and bird, flippers of seal and hands
structure consisting of humerus; radius-ulna bone
Organs because they have a common origin and similar basic for swimming, wingsof bats
Onganization but still they perform different functions like flipper of seal is used
and birds are used for flying.
writing, etc.
FOrelimbs of horse are used for running and hand of man is used for grasping things,
an ancestor which
ney have basic similarity in the structure of forelimbs because they inherited it from
had five
digited or pentadactyl limbs.
ne pentadactyl limb of the ancestral vertebrate became modified according to special needs of subsequent
and L0ns during evolution. Even we find homology/uniformity in arrangement of principal muscles, nerves
and blood vessels. Homology is based on divergent evolution.

Experiments 107
 Humerus

Radius-ulna

Carpals

Metacarpals

Phalanges
Human arm
Fore limb of bat (For grasping)
(For flying)
Fore limb of horse
(For running)

Humerus

Radius-ulna

Carpals
Metacarpals
Phalanges
Wing of bird
Flipper ofseal (For flying)
(For swimming)
animals
Fig. Homologous organs in

VIVA VOCE skeleton.

heart
 EXPERIMENT 30

AIM
isotateDNA from
plant tissues like papaya, pea seeds, spinach, etc.
To
RPOUIREMENTS shavings
Material: Any of the plant materials like papaya, pea seeds, spinach, onion, cauliflower
Biological.
plant.
leavesof flask, thermometer, ice cold
voung
or Glassware: Beeakers, test tube, measuring cylinders, glass rod, droppers, conical
mortar,funnel control, filter papers,
nestleand Test-tube stand, water bath, refrigerator/deep freezer with temperature procedure), blender.
Miscellaneous: (optional-depending on
balance. muslin cloth, blade, forcep, refrigerated centrifuge
weighing procedures.
Chemicals: As per different
it consists of twochains
THEORY forms genetic material in the most of the organisms and pairs.
DNA is the nucleic acid that spiral structure stabilized by hydrogen bonding between base
polynucleotides interwoven in form of
of isolation because:
shavings are one of the suitable materials for DNA
Cauliflower contains lot of
meristematic tissue and hence
) t has lot of
3
DNA. 5

no or little chlorophyll and less of mechanical

(i) There is
3.4 nm
tissue. membrane
Tannins are absent which otherwise keep the Sugar phosphate
(iüi) back bone
intact.
material because of: (A
Even onion is a suitable A-T base pair

(i) Its low cost long

1turn of helix (3.4 nm
(ü) Abundance -and has 10 base pairs)
(üi) Low starch content
(w) Lack of chlorohyll. -G-C base pair

PRINCIPLE

Isolation of DNA involves 3 steps: 5 3

(a) Homogenization Double Helical Structure of DNA
(b) Deproteinization
(e) Spooling plant tissue to break down cell. Heat treatment
blending present, would cut
Homogenization: It involves heating and denatures the DNAase enzyme which, if
L. the cell membrane and
BOnTens the phospholipids in not spool. cell membrane
DNA into small fragments sothat it willhomogenization media which breaks down the cell wall,
blender with
Tlant tissue is mixed in release of DNA. used to clean
and nuclear membrane, allowing the enzyme- Papain'-the common enzyme
protease easy to spool.
Deproteinization: It involves adding a clinging to DNA making the molecule flexible and
the proteins conc. (NaCI),
Alentact lenses. This denaturesdissociated from nucleic acids by high salt supernatant.
Alternativelyproteins can be
may be done to
remove particulate cell debris, leaving DNA in
Centrifugation
lomogenization medium includes: that breaks down cell membranes and
Dodecyl Sulphate) which is a biological detergent
uS (Sodium
Experiments 109
 emulsifies the lipids and proteins of the cell. SDS is a major ingredient in laund
can be substituted by Palmolive liquid detergent also.
(b) EDTA (Ethylene Diamine Tetra Acetic Acid) weakens the cell by binding the
Ca*) which are needed for membrane stability. This further aids in divale detecatrgeinotans S
nt
breaking
open the cells.
(c) NaCl (Sodium Chloride) enables nucleic acids to precipitate out of an alcohol solution

coalbeceasucse.e
the negative phosphate end of DNA causing the strands to come closer together and it s
III.
to stay inSpooling:
Precipitation of DNA involves adding ethanol to the solution which causes
solution except DNA. DNA will gather at the interface of solution and ethanol, and canevery
with a glass rod. be cOmpen
DNA fibres spooled on glass rod can again be dissolved in saline and sodium citrate and UV spoled
Z60 - 280 nm. Good DNA preparation gives 1.85 absorption. absorption recordet
PROCEDURE
Alternative methods are given to isolate DNA according to the suitability and availability of
Method I: chemicals.
Chemicals Required:
Palmolive liquid detergent, non-iodized sodium
juice of papaya or pineapple), 95% ethanol. chloride, distilled water, meat tenderizer or papain solution e
Preparation of solutions:
1. Prepare detergent-salt solution by adding l0 ml
2. Prepare 5% meat tenderizer solution by
detergent and 10g of salt to 90 ml distilled water
adding g of tenderizer (enzyme) to 95 ml distilled water. Jis
of papaya/pineapple (filtered through muslin
5
cloth) can be substituted for tenderizer.
3. 95% ethanol must be left in plastic
4. Prepare 5% NaCl solution by
container in the freezer overnight.
adding 5 g non-iodized salt to 100 ml distilled
Procedure: water.
1. To the plant tissue in 100 ml
beaker add 10 ml detergent-salt solution.
filter through muslin cloth. Homogenize in a blender and
2. To 10 ml of filterate, add 3 4 ml
meat tenderizer/papaya juice. Swirl the test
3. Carefully pour 10 ml ice cold tube to mix.
3minutes.
ethanol down the sides of test tube to form a
laver on top. Let it stand l
4. Using twirling motion of glass rod slowly more scored end of
collect mucus like DNA and glass rod through interface of two layers
place it in test tube with 5% NaCl
Method II: or distilled water.
Chemicals Required:
SDS. NaCl, sodium citrate, EDTA,
Tris HCl buffer, ice cold 95%
Preparation of Homogenizing Solution: ethanol.
1. Dissolve &-8 g of NaCl in 900 ml
distilled water. To
ml of 0.5 m stock of EDTA.
Make the volume to 1000this ml
add 43.7 g sodium citrate
and 50 g SDS
and?
2. TE Buffer
ma ml of 2 M stock of Tris
HCl pH 8.0 add 2 ml of 0.5 M
Procedure: stock of EDTA DH 8,0.
1 Heat. 100 ml
homogenizing
solution in a beaker in water bath
2. Dice 50 g of plant tissue/onion into small pieces and add it tountil
the itabove
reaches
solution.
60 °C. Let 1t standfor15
minutes.
3. Chill quicklyin an ice bath (15 20 °C) for 5
throughout. (This will slow down breakdown minutes
of DNA) swirl the solution gently to allow
even coolne

4. Homogenize it with blender for 30 sec at low

B. Filter speed and allow it to stand in ice bath for
15-20 minutes
through muslin cloth over a beaker in ice

110 7ogether witk Biology Lab Manual-XII

 some filterate into large test tube. Hold the test tube with filtered homogenate at an angle. Gradually
Pour
pourtwice the volume of ice cold 95% ethanol down the wall of test tube.
6.
precipitate out and spool it in one direction with the help of glass rod from the junction of two
DNA will
7. solutions.
Suspendthe DNA in TE buffer and store in freezer at (- 20 °C)
8.
MethodIII:
(CTAB Method)
most common and widely used method for DNAisolation from various plant groups.
Itis the
ChemicalsRequired:
older
(Cetyl Trimethyl Ammonium Bromide), NaCI, Tris HCl pH 8.0, EDTA, b-mercapthoethanol (for
CTABSDS (optional), chloroform, isoamyl alcohol, isopropanol, RNAase, sodium acetate, absolute ethanol.
or
leaves)
Preparation CTAB Solution
100 ml of 1 m Tris
20 g CTAB in 860 ml sterile double distilled water. To it add 81-82 g NaCl and
Dissolve
HClpH8,0and 40 ml
of 0.5 M EDTA pH 8.0. Autoclave and store at room temperature.
of

Procedure:
and mortar or liquid nitrogen.
, Crind 1-2 g of young leaf tissue in a prechilled pestle mercapthoethanol (for older leaves) or
SDS
ml of CTAB buffer (warmed to 65 °C) containing b
2. Add 10
(optional).
minutes to 1 hr.
3. Incubate at 65 °Cfor 30 temperature for
and add equal volume of chloroform: isoamyl alcohol (24: 1) shake at room
A Cool briefly
20 minutes.
15 minutes.
B. Centrifuge at 5000 rpm for
6. Decant aqueous phase
(top phase) into a new tube.
aqueous phase and mix gently.
7. Add 10 ml isopropanol to 500 ml ice chilled
10 minutes. Decant isopropanol and wash the pellet with
8. Centrifuge at 5000 rpm for
dry.
90 % ethanol and then drain double distilled water to dissolve
the pellet.
TE buffer or sterile
9. Dissolve pellet in 100 ml

ozSERVATION ice chilled ethanol.

junction of two layers on adding
Shiny white DNA is seen at

and the
cells. The chromosomes were broken in the process
DNA found in plant
This DNA represents all the treatment.
chemical
DNA precipitated due to

FRECAUTIONS make the solutions.

1. Use distilled water to
make solutions carefully.
2. Weigh the chemicals and
freezer overnight in plastic bottles.
3. Ethanol should be kept in
VIVA VOCE
DNA extraction?
Q.3. What is the use of EDTA in
Q1. Who gave the structure of DNA? Ans. EDTA binds to cations in
the cell which
used to
Ans. Waston and Crick. maintain the membrane str. Hence its
Q.2. What was the structure based on? break open the cells.
Ans. Complementary base pairing rule and X-ray Q.4. Which kind of tissues are rich
in DNA?
crystallography. Ans. Meristemnatic tissues.

Experiments 111