Objective: Human cell (Cheek Cells) Isolation, Staining and Observation under Microscope.

Learning outcomes:
1. Mount and stain a specimen on a slide and prepare it for microscope viewing.
2. ldentify parts of a compound microscope and operate it effectively.
3. Understanding of basic structure of animal cell.

Principle: As in all animal cells, the cells of the human cheek do not possess a cell wal. A cell membrane
that is semi-permeable surrounds the cytoplasm. Unlike plant cells, the cytoplasm in an animal cell is
denser. granular andoccupies a larger space. The nucleus is present at the center of the cytoplasm. The
absence of a cellwall and a prominent vacuole are indicators that help identify animal cells, such as cells
seen in the human cheek. The cell has different parts, can absorb stains or dyes are referred to as
chromatic. Without stains, cells would appear to be almost transparent, making it difficult to differentiate its
parts. Methylene blue has a string affinity for both DNA and RNA. When it comes in contact with the two, a
darker stain is produced and can be viewed under the microscope.

Requirement: Microscope slides, Cover slips, Methylene Blue solution, Dropper, Saline (0.9%) solution,
Blotting paper/Tissue paper, Microscope, Toothpick.
Procedure:
1. Place one drop of Saline solution on the cleaned glass slide.
2. Gently scrape the inner side of the cheek using a toothpick, which will collect some cheek cells.
3. Mix the saline solution and the cheek cells usinga tooth pick.
4. Take a few drops of Methylene blue solution using a dropper and add this to the
slide. Make sure that cells are getting sufficiently stained. mixture on the
5. Take aclean cover slip and lower it carefully on the mixture with the aid of a
6. Remove any extra stain or liquid around the cover slip using a needle.
7. Place the glass slide with coverslip on the stage of the blotting/tissue paper.
compound microscope and observe the cells
under 10X and 40X objective lens.

Obserrations:
1. A large number of flat and
2.
irreqular-shaped cells are observed.
The cells do not have a cell wall. However, each cell has a thin
cell membrane.
3. A deeply stained nucleus is observed at the center of each cell.
4. No prominent vacuoles are observed in the cells.

Resultlnterpretations: The nucleus at the central part of the cheek cell contains DNA. When a drop of
methylene blue is introduced, the nucleus is stained, which makes it stand out
the microscope. The entire cell appears light blue in and be clearly seen under
color; the nucleus at the central part of the cell is much
darker, which allows it to be identified.

nuclear
membrane cytoplasm

cell/ nucleus
Anembrane

Figure A and B: [A]Schematic digram of Human cheek cells and

[B] Appearnce of cells under microscope.
Precautions:
1. Ensure toothpick used to scrape the
2. Extrastain should be removed using cheek clean, so it does not cause infection to the cheek.
is
3. Discard the toothpick in Sodium blotting paper.
hypochlorite solution and glass slides in beaker containing the
disinfectant solution after the completion of experiment.
Aim : Human cell DNA isolation and quantification.
Material Required:Sodiunm Chloride(0.9"%), Delergent solution (nebulin, 12-15º%). Isopropanol(ice coldy95%
Ethanol,MicroCentrifugetube (1.5m),Centrifugetube (50ml)and Dropper.

Theory: DNA (deoxyribonucleic acid) is the genetic material in living organisms but some viruses contain
RNA as genetic material. DNA is the Blueprint for Life, found in the nucleus of eukaryotic cells and
nucleoid of prokaryotes which contains the necessary infomation to carry out all the living processes of the
[Link] isolated DNA can then be used for downstream applications such as PCR, sequencing, or cloning.
Currently, it is a routine procedure in molecular biology or forensic analyses. The basic principle behind
DNA isolation is disruption of the cell membrane, and nuclear membrane to release the highly intact DNA
into solution followed by precipitation of DNA and removal other contaninating biomolecules such as
protein and lipids .The specific role of most common chemicals used for DNA extraction include:
1. Salt, such as NaCl, which is often used to help in neutralizing the DNA (the positive sodium ions (Na
present in NaClarc attracted to the negative charges of the DNA's phosphate )and makes it less hydrophilic
i.e less soluble in [Link] it also also helps in detaching protein molecules from strands of DNA and
makes the extraction process casier.
Detergents, such as SDS or Tween-20, which are used to break open cells and release the DNA into the
solution.

3. Chilled isopropanol or cthanol, which arc uscd to prccipitate the DNA and allows it to flocculate and
settle very easily and quickly.

A. Protein

Head
Celt menbrane Lipld
Tail

DNA

ouclear
membrane
Enlarged view of coll membrane.
Nuclear memnbrane is the same.

Cell Detergent

urel:
cell using (A) Enlarged
detergent view of eukaryotic cell membrane. (B) pictorial representation of disruption o
Procedure:

A) Obtaining the chcek cells

1. Take a drink of 5 ml. Sodium Chloride solution (0.9 %) and swirl it for Imin around in your
mouth (without swallowing) and then spit the solution into the 50 ml centrifuge tube.
81ub93o1
B) Releasing the DNA from inside the cheek cells
issdO(/
1. Add [Link] 12-15% liquid detergent solution to the "cheek" mixture in the centrifuge tube. EI
2. Plug the cap into a centrifuge tube and gently invert mix the contents of the centrifuge tube, 5
times. Do not shake the centrifuge tube & wait for 5 minutes. e6sl91 (
n2 bbA.I
C) Precipitate the DNA Brli gul9.C
Tilt the centrifuge tube carefully add Sml. of isopropanol (ice cold) and invert mix the solution 23
times. Hold the centrifuge tube in an upright position for 2-4 minutes. You will see a spindly white
substance, maybe clumps of it forming on top of the salt/detergent mixture. iqiT9 (
3939rlj tliT
D) Collect the DNA
gnqun6 ni
Use the dropper to collect the DNA and transfer it into a fresh 1.5 ml .airnmot ti to
do this very carefully as the DNA is fragile.
microcentrifuge tube, you must
s 139lloD (0
Result: A white or milky precipitate of DNA will be observed on the sides or bottom of centrifuge tubes
after precipitating with iso-propanol.
ob j2urn wy
Precaution

1. Always wear gloves while handling

[Link] liquid contaminates in sodium hypochloride solution9)
while dispose contaminated gloves, centrifuge tube and dropper in biohazard dustbin.
2. Avoid vortexing during DNA extraction.
otusT919
3. Solution such NaCl should be
autoclaved before using during experiments.
9volg
 OBJECTIVE: PERFORM AGAROSE GEL ELECTROPHORESIS FOR SEPARATION &
DETECTION OF DNA MOLECULES
Principle: Electrophoresis is a technique to separate charged molecules in an electric field.
Useo to separate DNA, RNA according to their Agarose gel electrophoresis I5
molecular weight. This is achieved by moving negatively charged nudeic ecid
oecuies (negatively charged due to their phosphate backbone) through an agarose matrix with an
OT the gel pores can be controlled by simply adiusting the electric field. The size
separation of a wide range of different-size nucleic acid
agarose concentration to prepare gels appropriate ror the
malecules. Small DNA molecules move more quickIy
than larger
DNA molecules. Agarose gel electrophoresis is the most tnoug the gel
Sizes ranging from 100 bp to 25 kb. This technique effective way of separating DNA fragments of varying
is used for estimation of the size of DNA mnolecules
enzyme digestion, e.g. in restriction mapping of cloned DNA, analysis following restriction
of PCR products, e.g. in molecular diagnosis or genetic
fingerprinting.
Agarose, which is extracted from seaweeds genera Gelidium and Gracilaria, is liner
polymer of D-galactose
galactose. Agarose gel are cast by boiling agarose in presence of a buffer, then poured and 3,6-anhydro
to form a matrix. The most into a mold and allowed to harden
common dye used to make DNA or RNA bands visible for agarose gel
bromide (EtBr). It give pink/orange fluoresces under UV light when electrophoresis is Ethidium
intercalated
fluorescent nucleic acid gel stain, is a superior DNA stain that is nontoxic and
into DNA & RNA. SYBR Green I, another
sensitivity as ethidium bromide. non-mutagenic and has similar DNA detection

Side vicw: Sample loded

into well

Plastic gel bos

Bufler

Elctrie feld and

Dircction of migration

Negative (-) Electrode Positive (-)Electde

Factors affecting electrophoresis activity:

a- Agarose concentration
b- Applied voltage
-Electrophoresis buffer (TAE or TBE)
d Ethidium bromide effects

Materials Required:
Equipment: Vortex shaker, pH Meter, Weighing balance, Gel documentation system, Cel casting plate,
with Power Supply Electrophoresis unit
Glassware: Beakers, Reagent bottles, conical flask, measuring cylinder
Chemicals: Agarose, Ethidium bromide, loading dye, TAE buffer and DNA samples
Other requirements: Micropipette, Micropipette Tips, Eppendort tubes, Gloves

Preparation
1-Ethidium bromide: 10 mg. Iml. in H,o
2- Sample loading/Tracking dye: It contains two
different dyes (bromophenol blue and xylene cyanol FF) for visual
of DNA migration during electrophoresis. The
presence of glycerol ensures that the DNA in the ladder and samole tracking
layer at the bottom of the well. Bromophenol blue o.25, Xylene forms a
cyanolo.25%,
3- Electrophoresis buffer (TAE 50X ,pt 8.0) Tris Base: 242 gm, Clacial
Glycerol 5o%, in 1X TAE buffer
Acetic acid: $7.1 ml, EDTA (o. 5M):100 ml., make final
volume 1000 ml. with help of distilled water.
Procedure:
1. Take1 gm of Agarose in a 500 ml conical flask. Mix it with 100
ml 1X TAE buffer and then heat in a microwave oven or on
hot plate until completely melted.
2. Let the solution cool down to about 50C at room temperature, or water bath. Stir or swirl the solution while coolnd
Note -Now wear gloves when handling Ethidium bromide. (EtBr is a mutagen)
 3. Add t0 l Ethidium bromide per t00 ml gel solution for final
concentration of 1 ug/ml
4. Stir the solution to disperse the Ethidium bromide. Then pour it
into the gel rack/casting tray.
5. Insert the comb at one side of the gel, about 5-10
mm. from the end of the gel.
b. When the gel has cooled down and become solid, carefully remove the cob. The holes that
as the wells/slots. are remain in the gel, known
7. Mount the gel, together with the rack/tray, into
electrophoretic tank containing 1XTAE
8. The gel must be completely covered with TAE, with the
wells/slots at the end of negative electrode.
9. Mix the sample of DNA with gel-loading buffer (eg. Mix 5ul
DNA sample with tul gel loading butfer).
10. Load DNA samples mixed with loading buffer slowly into
the wells using micropipette.
11. Apply DNA Ladder mixed with loading dye beside the sample
well.
12. Close the lid of the gel tank and attach the electrical leads to the power pack so
that the DNA will migrate toward the
anode and apply a voltage of 5- 10 V/cm.
13. Confirm the flow of current by observing bubbles coming
off the
t4. DNA will migrate towards the positive electrode (anode) which is clectrodes.
usually coloured red.
15. Run the gel until the DNA sarmples or dyes (xylence cyanole) has migrated
the appropriate distance through the gel (- 40
60 minutes), turn off the electric current and
removes the electrodes nd cover from the gel tank. 16. Exarmine the gel on UV
transilluminator or Gel documentation system.

Observation: Report the observation from the image obtained after documentation.
Result: DNA fragments are separated in electrical field according to Tolecular
weight. The result is a series of 'bands', with
each band containing DNA maleccules of a particular size. The
bands furthest from the start of the gel contain the smallest
fragments of DNA, The bands closest to the start of the gel contain the largest DNA
fragments
Reference: Sarnbrook, J., Fritsch, E.F. and Maniatis, T. (198g) Gel electrophoresis of DNA. In:
Maniatis, T. (Eds.) Molecular Cloning: a Laboratory Manual, New York: Cold Spring Harbor Sambraok, J., Fritsch, E.F. and
Harbor, NY, USA Laboratory Press, Cold Spring

NEGATIVE ELECTRODE
,Electric
Curent
Plasmid 0333 DNA Bands
Vector are
DNA
separated
By
Size
MOLECUARSTATION
Add DNA Sample onto
Agarose Gel Lane #2
(DNA Ladder is in Lane #1) POSITIVE ELECTRODE

DNA Bands are

Exposed on Film

1kt

Dye Added S00 bp

Binds to 200 bp Under UV
DNA Light DNA Is
Copyr1ght 2008 Molecular [Link] Visible
 Aim: Biosynthesis or Green Synthesis of Gold Nanoparticles using leaf extract of Neem
(Azadirachta indica).

Principle : Nanoparticles (NPs) have multiple applications in Biology and Biomedical field, These particles can
be prepared by different chemical, physical and biological approaches. Biosynthesis or Green Synthesis of gold
nanoparticles using neem leaf extract involves a bottom-up approach utilizing the phytochemicals present in
neem leaves to reduce gold ions (Au*) to gold nanoparticles (Au). The process is often considered asimple and
eco-friendly approach to nanoparticle synthesis. The phytochemicals present in Neem are namely terpenoids
and flavanones, which act as reducing as well as capping agent and helping in stabilizing nanoparticles. The
nanoparticles can characterized by various techniques UV-VIS spectroscopy, FTIR, XRD, TEM arnd FESEM. Gold
nanoparticles' interaction with light is strongly dictated by their environment, size and physical dimensions.
Small (~30 nm) monodisperse gold nanoparticles, the surface plasmon resonance phenomenon causes an
absorption oflight in the blue-green portion of the spectrum (-450 nm) while red light (-700 nm) is reflected,
yielding a rich red color.

Requirements:
Reagents: Gold Chloride solution (HAuCl.3H,0) -1mM, Neem Leaf Extract, DIWater
Equipment: UV-visible Spectrophotometer, Hot plate cum stirrer
Others: Glass beaker, Micropipette and tips

Procedure:

1. Preparation of Neem Leaf Extract (Reducing agent)

Neem leaves were collected,washed thoroughly and air-dried. 40 g of neem leaves were boiled in 50 ml of DI
water for 15 mins. The solution iscooled down and filtered to obtain the neem extract.

2. Preparation of Gold Nanoparticles

Take 100ål of Gold chloride solution (Precursor solution) from.1 Mstock solution and add 9.9 ml of DI water in
a beaker. Let the 10 ml solution heat to 150°C while stirring at 500 rpm for 10 minutes. On boiling add 100ul of
prepared neem extract dropwise. The reaction should stop/remove the beaker on noticing acolour change from
yellow to red wine. The solution will be used directly to characterize using UV-visible spectroscopy by recording
the absorption spectrum.

Observation: The yellowish solution turns red wine red, indicating the formation of gold nanoparticles. The
gold nanoparticles show maximum absorbance in the 520-540 nm range.

Conclusion: Green metal nanoparticle synthesis has been successfully conducted using anatural source.
Aim: Separation of plant pigments by Paper chromatography

Principle: Chromatography is a technique used to separate moleules on the basis ot differenes in

size, shape, mass, charge. solubility and adsorption properties The term hromatography is
derived from Grek words Chrom-tolour and Gtaphe-write There are many types of
hromatography aper chonatography, column chromategraphy. thin laye chromatography
and patition chromatograph These tchnques volve the interaction between three
components the mixture to be separatenl, a olid phase and a solvent
Thotosynthetic plants convert light energy trom the sun to chemical foxod energy. During
photosynthesis, molecules referred to as pignents are ud to capture light energy. I'igments ar
chemkal compounds whch retlect only certain wavelengths ot vIsible light. Plant leaves contain
four primary pigments chlorophyll a (dark green), chlorophyll b (yellowish-gren). xanthophylls
(vellow) and carotenoids (orange).
In paper chromatography. the mixture is spotted onto the papr, dried and the solvent is allow ed
to tlow along the sheet by capillary attraction. As tlhe solvent slowly moves through the paper, the
ditterent compounds of the mixture separate into different coloured spots The paper is dricd and
the positon of different compounds is ViSualized. The prniple behund the papeT chromatography
Is that the most soluble substaces move further un the tlter paper than the least soluble
substances. Different plant pigments can be separated by using the technique of paper
chromatograpl1y.
Materials:

Fresh leat sanples, Mortar atud pestle Chromatgraplhy paper, Beaker. Sissor, Pencil and Sale
Capillary tube, aluminium foil
For crusing ot leaves use - Ethanol or Acetone- 5 to 10 mL only
Solvent System is I'etrolum ether Acctone 1

Procedure:

1, lake a few fresh yreen leaves and using, scissor cut the leaves into small pieces.
2. By usng
us (5-10 ml) Ethanol /Acetune grind the eaves
a Chromatography apr and with help of a pencl, scale and draw a horizontal line
about 1-2 cm awav trom the one end of pape.
I'ut a drop of the pigment extrat in the middle of the line with the help of a capillary tube
Allow the drop to dry and repeat till three or tour tumes until a concentrated spot is obtained
Take the chromawgraphic chamer/beaker and pour ether acetone solvent in it
Fold the hromatogaphy papur and staple it's both the ends
The oadn spot should reman about lcm above the solvent level.
Leave the chromatogaphu chamnber/beaher undisturbed for some time (10-20 minutes).
10 Oberve, as the solvent moves through the paper. it separate the ditferent pigments of the
misture to various distances.
[Link] the solvet rises about 3/4th up to paper, remove the paper carelully and let it dry Not
the distance travel by solvent
12. Calculate the Rf value usiny, the eyltation and reord the vaiues in the observation table.
Observation: The dried chromatugraphc paper strig shows four distint paper bands Dtferent
Piments can be identified by their colours,
Colour R{ Value |Pigment

Calculation:

R{ Value of the each pigment spot can be calculated by the equation.

Rf= Distance travelled by the pigmen/Distance travelled by the solvent

Measure the distance of each pigment band from the loading spot and also the distance travelled
by the solvent.

Result:
Each pigment has individual Rf Value
The topmnost orange yellow band of p1gments in the separation corresponds to carotene.
The vellowish band appearing below it indicates the xanthophylls,.
The third from above dark green band represents chloroplhyll a
The lowermost yellowish green band is that of chlorophyll b
Irecaution:
Leaves should be fresh and green.

While hanging the paper in the chromatography chamber/ beaker, the oading spot should remain
about 1 cm above the solvent level

Solvent is flammable and handle with care

Cover the mouth of beaker with aluninium foil

Staple the chromatography paper correctly otherwise it interferes in movement of solvent.

Do not inhale the solvent, it may be toxiC.

Solvent front should mark immediately.

 protein and
Aim: Toidentify the presence of common biological macromolecules (carbohvdrate.
lipid).

P'rinciple: Living organisms are composed of four muior classes of macromnolecules: Croony I
Lipids, Proteins and Nucleic acid. They have diverse shapes and sizes and serve a variety of pap
this laboratory you will investigate three classes of macromolecules: carbohydrates (reoues lecules
starch), lipids, and proteins. We willuse qualitative chemicalindicatorsthat tum color if certain molve will
are present. In order to understand itfa certain color indicates a positive result or ancgative resu
run a positive control and a negative control for each test. The positive control will show what a posie
result should look like; a negative control will show what ancgative result should look like

Test Proccdure Observation image

Name
Requirements:
0 lodine solution Add 10gm
potassium iodide in 200 ml water.
add 6.5 gm iodine, andstir to BrnTrc
dissolve make tinal volume up to
500 ml.
O Store in a dark colour bottles.
D Micropipette. tips, test tubes, test
solutions (Potato. Juice. Egg fr Neathe Sarch Abent fet Fosv Sarh Pet
white. W hole milk &
vegetable oil), *ve controls, -ve Figurel. lodine test for starch
controls and droppers.
Procedure
1 Designate the est ions
control, ve control.
Potatato, . Fgg white, Whole milk
& vegetable oil)
Add 2ml of water to the "-ve
2
control" test tube
Add 2mL, of Starch solution to the
*ye control" test tube.
Add 2ml. of test solution
Add two drops of iodine to cach test
tube
Record your observations:
Positive Result: Appearance of deep
blue black colour - indigates presence of
starch.
Result Appearance
Negatise indicates absence
brown vellow colour
of starch
 A
Requirements:
O Fehling's Solution A 35 gm of 2Cu

[Link],O in 500 ml. of water Fchling's

solution
D Fehling's Solution B- 120gm of
KOH & 173 gm of sodium
Test) potassium tartrate in S00 [Link]
water. Mix both in equal volumes
(Fehling's just belore use. Carhoxylate
C'u0
Red-hrown
0 Micropipette. tips, test anion
tubes, test
solutions, tve control, -ve control
& water bath
Sugar) B
Procedure Fehling's Test
(Reducing 1. Designate the test tubes: ve
control, tve control, test solutions Beathe
(Potato, Juice, Egg white. Whole milk &
vegetable oil).
e 2. Add Iml of water to the "-ve
control" test tube.
3. Add ImL. of glucose solution to the
*+ve control" test tube. Figure2 A) Chemical reaction involved in
4 Add Iml. of test solution and Iml
Febling's Test. B)
Fehing's test for sugars
Fehling's solution in each test tube.
5. Mix well and place test tube in
boiling water bath for 4-5 minutes.

Record your abservations:

Positive Result: Appearance of reddish
brown precipitate indicates presence of
carbohydrate.
Negative Result: Appearance of a deep blue
color indicates absence of rcducing
sugar
Cquiements
Water, Sudan ll reagent, vegetabie oil.
Micropipette and tips. test tubes. test
solutions (Potato, Juice, Egg white, Whole
milk & vcgetable oili and control solution,
Proceture
1 Designate the test tubes: -ve
control, ve control, test solutions
Juice, Egg white, Whole milk
egetable oil)
Add iml of water to the
cantrol" test tube
Add 2nl of vegetable oil to the
*ve control" test tube. lest tiilts for fats
Iueure 4, Sutan
4 Add 2ml. of test sotution and 2mt
water soultion in cach test tube
Then add Sdrops Sudan
cach test 1ube
reagent, mix well in cae
Record your observations:
Positive Result Appearance of ted
colourcd comples on top indicates
presence of fats
Negative Result: Appcarance of uniforn
red colour- indicates abscnce of fats.
 Requirements: Brown paper, Water. Oil.
foods to test
Procedure:
IWrite your name on the brown paper
test) 2 Fold the paper into 5 sections
3. Label the sections -ve control, *ve
paper control, Food #
4. Place one drop of waler on the- control"
(Brown [Link] it into the brown bag.
s Place one drop of oilon the "* control"
section
Lipid/Fats 6. Gently add asmall amount of Food # on
the Food #" seetion.
8 IMPORTANT: Wipe away excess food
with paper towel into the trash"
for 9 Set aside to dry
Test
10. At the cnd of the period. compare cach
food smear to your controls
Observations:
0 Brown paper is dark and translucent
(see-through) ’ indicates presence
of fats The paper becomes
translucent (allows light to pass
through) in the presence of fats.
OBrown paper stays brown -
indicates absence of fats
 Requirements:

40°, NaOH. 1°% CuaO4, Micropipette and -Hlue Coko

Purple Colg
(Protens Preserti
tips, test tubes, test solutions. (Protens Atse)

Procedure Positive Biuret Test

Negative Biuret Test
1 Designate the test tubes: -ve
test)
tRioret
Protein
control, *ve control, test solutionsigure3. Biuret test results for protein
(Potato, Juice. Igg white. Whole milk &
cgetable oil).
2 Add Iml of water to the "-ve
control" test tube
Add Iml. of peptone solution to the
*ve control" test tutbe
Add Iml. of test solution and Iml
4,NaOH soultion in cach test tube.
Then add 5-8 drops of I" CuS04,
m well
Record your obervations:
Postne Result Appearance
of
puple p1nk violet colourcd complex
ndacates the prescnce of protein
bcyait Reuit Appearance of blue colour
ndicates atncnce of ptoen
 Student Data and Analssis
Te Pacean in he boifygou belhevc the fod wil cent
ihis fnl itet

Mik

Eg
Vegetable ol