Shri Dhaneshwari Manav Vikas Mandal’s

(S. D. M. V. M’s)

College of Agricultural Biotechnology

READY-471
In House Skill Development Module
(Molecular Biology)

Speaker
Mr. Pawankumar Kharate (Asst. Prof.)
Ph.D. in Agriculture (Submitted)
Plant Molecular Biology and Biotechnology
 Practical-1: Equipment and Instruments

Aim :- To familiarize with equipment and instruments used in

molecular biology lab

Autoclave: for sterilization of

solutions, media, and other
lab wares. Temperature at 121
°C, pressure 15 lbs and time
15 – 20 minutes

Dry bath: for incubating

solutions at specific
temperatures
Water bath: for
heating solutions
and DNA samples at
a specified
temperature

Incubator shaker:
for incubating
bacterial cultures or
hybridization filters
at a constant
temperature and
speed
Centrifuge: for spinning
and reprecipitating DNA
samples at low speed
(2,500-12000 rpm)

Microcentrifuge: for
spinning and re-
precipitating small
amounts of DNA samples
at higher speed (10,000-
14,000 rpm)
Inoculation cabinet/
Laminar Flow Hood:
clean air hood for
aseptic preparation of
cultures

pH meter: for adjusting

or measuring pH of
solutions and reagents
Micropipettes: for
accurate dispensing of
DNA samples, solutions,
and reagents

Freezer (-200C and

-800C) : for storage of
restriction enzymes, DNA
samples,
solutions/reagents and
other chemicals that
require low temperature
storage
Quick ice maker: for
quick ice making required
in various molecular
biology procedures

Thermal cycler machine:

for amplifying DNA
samples
Agarose gel electrophoresis
system: for separation of
fragments of DNA and proteins
on agarose and poly
acrylamide gels

Microwave oven: for heating

solutions or melting agarose
gel
Milipore system: to obtain
ultra pure water.

U. V. Transilliminator/ Gel
Doc: for visualizing DNA
samples on the agarose gel.
Nano Drop spectrometer: for DNA,
RNA, protein quantification
Additional equipments
 Mortar and pestle: for grinding leaf tissues
in liquid nitrogen.
 Thermo flask: for storing and dispensing
small amounts of liquid nitrogen (usually 1.0
– 2.0 L capacity).
 Liquid nitrogen tank: for storing large
amount of liquid nitrogen, usually 20L
capacity.
Other supplies and instruments commonly used
1. Forceps
2. Scissors
3. Scalpel
4. Disposable gloves
5. Paper cutter
6. Paper towels
 Ice bucket
 Petri dishes
 Eppendorf/
microcentrifuge tubes
 Tips
 Labeling/Colored
tapes
 Centrifuge tubes
 Marking pens
(assorted colors)
 Reagent bottles
 Beakers
 Flasks
 Test tube
Result
 The uses of equipments and instruments
of molecular biology lab was successfully
examined
 Practical-2: DNA Extraction
Aim : To study the isolation of plant genomic DNA by
using modified CTAB method
 Basic Principle

1. Extraction of high molecular weight DNA free from protein and

RNA is essential for all molecular biology investigations.
2. The cell walls must be digested away to release cellular
constituents, which is done by grinding tissue in extraction
buffer or with liquid N2.
3. Detergents like CTAB (Cetryl Trimethy Ammonium Bromide) or
SDS (Sodium Dodecy Sulphate) are used to remove
biomolecules and protein other than nucleic acids.

 CTAB can bind with the cell wall polysaccharides and protein and by
complexing with it.
 It co-precipitates the macromolecules along with the DNA which is removed
by the centrifugation, later on.
 Basic Principle
4. DNA must be protected from the endogenous nucleases and
therefore EDTA (Ethylene Diamine Tetra Acetic Acid), a
chelating agent is used. It binds to the magnesium ions (Mg+
+
) which are considered as a co-factor for most nucleases.
5. The tissue mixture is emulsified with either chloroform :
Isoamyl alcohol to denature protein from DNA.
6. The crop species where excess polysaccharides/phenols are
present PVP (Poly Vinyl Pyrrolidone) is added to the
extraction buffer.
 Upper aqueous phase: DNA, salts and sugars
Inter phase: Denatured proteins (white fragment of lysed protein)
Organic phase: Lipids, broken down proteins and other cell debris
 Chemical required Role
TRIS – HCL (1M) : Maintains pH
EDTA (0.5M) : Chelating agent
CTAB : Remove biomolecules and protein
SDS : Remove biomolecules and protein
Sodium Chloride : Helps in DNA precipitation
β- mercapto ethanol : Cell lyses and denatures proteins
Potassium Acetate : Helps in cell debris precipitation
Sodium Acetate : Helps in DNA precipitation
Ethanol (pre-chilled) : DNA precipitation
Isopropanol (pre-chilled) : DNA precipitation
Chloroform and Isoamyl alcohol : Denature protein from DNA
RNase A : Degradation of RNA molecules
Labwares required
1. Scissors,
2. Disposable gloves,
3. Paper towels/Kimwipes,
4. Ice bucket,
5. Micro-centrifuge tubes,
6. Micro pipettes and
7. Micro pipettes tips
(1000 µl, 2- 20 µl, 0.5 - 2 µl)
8. Labeling/Colored tapes
9. Reagent bottles (500 ml/200ml)
10. Marking pens
11. Beakers
12. Eppendorf racks
13. Tissue paper rolls
1M Tris Stock
 Weight out 121.1g of Tris base [Tris (hydroxymethyl) aminomethane ; MW 121.1g/
mole] and place into a 2000 ml beaker containing a magnetic stir bar.
 Add 800 ml of distilled water and dissolve the Tris with stirring.
 Adjust the pH to the desired value by addition of concentrated hydrochloric acid
(HCl).
 The addition of HCl should be done carefully, while the solution is stirring and being
monitored for pH, to avoid a drastic drop in the pH.

0.5 M EDTA stock

 Weight out 186.1 g of disodium EDTA (disodium ethylenediamine tetra
acetic acid, dehydrate; MW 372.2 g/mole) and place into a 2000 ml beaker.
 Add 800 ml of distilled water and stir vigorously, using a stir bar and a
magnetic stirrer.
 Adjust the pH to 8.0 using concentrated sodium hydroxide (NaOH) to
facilitate dissolution of the EDTA.
Components CTAB extraction buffer
CTAB - 20 gm
1M Tris-HCl (pH 8.0) - 200 ml
0.5 M EDTA (pH 8.0) - 100 ml
5M NaCl - 400 ml

Adjust pH to 7.5 with concentrated HCl

Procedure
1. Fresh leaves are cut into small pieces, transfer into 1.5 ml tubes
and 400 µl CTAB buffer. Crush the leaves in tissue lyzer (with
bead for 1 – 3 min.).

(Alternatively leaves can be crushed using liquid nitrogen using mortar

and pestle and transfer immediately into a 15ml/50 ml centrifuge tube)
2. Add 400µl of CTAB extraction buffer (preheated to 650C) in each
grounded sample and mix thoroughly.
3. Incubate samples at 65°C for 15 – 20 minutes using a water bath
with occasional mixing.
4. Add equal volume (~700 µl) of choloform:
isoamylalcohol (24:1) and shake slowly.
5. Keep at room temperature (~25°C ) for 15 min. and
shake slowly in between.
6. Centrifuge samples for 3 minutes at 13,000 rpm.
7. Two aqueous layers separated by a whitish thin layer
will be obtained.
8. Transfer the aqueous phase (~500 µl), without
disturbing inter-phase, into a fresh 1.5 ml tube.
9. Add equal volume of pre-chilled absolute ethanol
(~500 l) to each sample to precipitate the DNA.
Thread like white DNA will appear to precipitate.
10. Keep it at -20°C for 2 hours in deep freezer.
11. Centrifuge at 10,000 rpm for 7 – 8 minutes to
obtain transparent white DNA pellet. Pour off the
ethanol from the samples and allow it to air dry
for 2 hours (in laminar air flow) to remove the
ethanol completely.
 
12. Wash the DNA pellet with 70 % ethanol, by short
spin for a minute followed by draining ethanol.
13. Suspend the DNA pellet into 50 l of TE buffer
(10mM Tris: 1mM EDTA, pH 8.0) and store at - 20C
till further use.
14. The DNA so obtained has to be quantitatively and
qualitatively evaluated before any application.
Results:
1. Thread like DNA start appearing with addition of pre-
chilled ethanol/iso-propanol in respective stage.
2. DNA pellet of transparent to white colour is obtained
after final centrifugation
 Practical-3
Quantitative estimation of nucleic acid
Aim : To study the quantitative estimation of nucleic acid
with the help of Nanodrop Spectrophotometer.
 Basic Principle
Nucleic acid (DNA and RNA) has maximum
absorbance at about 260 nm.
The ratio between the readings at 260 nm and 280
nm (OD 260/OD 280) provides as estimate for the
purity of nucleic acid.
Pure preparation of DNA and RNA has a ratio of
approximately between 1.8 and 2.0.
If there is contamination with protein or phenol the
ratio will be significantly less than 1.8 (< 1.8).
A ratio greater than 2.0 (>2) indicates a high
proportion of RNA per DNA in the sample.
 Procedure
1. Start the nanodrop spectrophotometer and set
wavelength at 260 nm for DNA
2. Gently clean the sample feed point or lower
measurement pedestal with DDW.
3. The 2 µl of TE buffer was loaded on to the lower
measurement pedestal (Nano Fall).
4. Then sampling arm (lid) was lowered into the
‘down’ position to set the 1st blank reading with a
range of 0.0 to 0.1.
5. After the blank reading buffer was wiped from
both the pedestals using tissue paper or blotting
paper.
6. Then 1 or 2 µl of DNA sample was loaded on to
the lower pedestal (Nano Fall) and reading
showing the quantity of DNA was recorded on
nanodrop spectrophotometer.
7. Both the measurement pedestal surfaces should
cleaned with 4 µl of autoclaved distilled water and
gently wiped with tissue paper before loading of
other DNA samples.
 Results:
Nanodrop reading:
Quantity in nanograms : 3632.4 ng/µl
Ratio (260nm/280nm) : 1.81
1. The concentration of DNA in given sample was
3632.4 ng/µl which is in good amount and further
dilution on this sample is required for PCR
amplification
2. The DNA of given sample is free from RNA and
protein contamination on the basis of readings at
260 nm and 280 nm OD
 Upon quantification, the DNA was diluted with TE or
sterile water (nuclease-free water) to make final
concentration DNA upto 50 ηg/μl for PCR amplification.
 Dilution was carried out according to the following
formula:
N1 V1 = N2 V2
 
Where, N1 is the concentration of starting solution
V1 is the volume of starting concentration (?)
N2 is the concentration of final solution
V2 is the volume of the final solution

3632.4 X V1 = 50 X 1000

V1 = 13.76

Finally take the 14 μl of DNA sample and add into the 986 μl
of TE or sterile water (nuclease-free water) for PCR
amplification.
 Practical-4
Qualitative estimation of genomic DNA

Aim : To study the qualitative estimation of DNA with the help

of agarose gel electrophoresis.
Basic Principle
 It is critical that plant DNA must be of high molecular weight for
molecular analysis (> 30 kb).
 The isolated DNA needs to be estimated for quality before starting of
molecular biology experiments.
 To check this you will need to run an aliquot (2l) of each sample on
0.8 % agarose gel.
 High molecular weight DNA will reveal single band near high
molecular weight size band of molecular weight standard.
 If a second band is revealed at the lower portion of the gel, it
indicates contamination of RNA and a smear in the gel shows protein
contamination and degraded DNA.
 A further purification is needed according to DNA quality
observation if necessary.
 High molecular weight and high quality DNA can be used for all types
of molecular studies
Chemicals required
• Tris (1M)
• EDTA (0.5M)
• Glacial Acetic Acid
• Autoclaved distilled water (ADW)
• Standard DNA
• Ethidium Bromide
• Ethanol (70%)
• Agarose
• DNA loading dye (6X – orange loading dye)
• TAE (1X) buffer
Material : Genomic DNA
Labwares required
 Agarose gel electrophoresis unit with power pack,
 Conical flask,
 Micropippetes and tips.
Apparatus required
 Gel Documentation system
Preparation of Reagents
10X TAE
TAE Buffer – pH 8.2 1000 m

Tris Base 242 g

Glacial acetic acid 57.1 ml
0.5M EDTA (pH 8.0) 100 ml
* Finally makeup volume to 1000ml

1X TAE
Prepare 1X TAE as working solution from stock of 10X
TAE by taking 10 ml of 10X TAE and 90 ml double
distilled water (DDW)
Preparation of 0.8 % agarose gel
1. Take 100 ml 1X TAE buffer in a conical flask
2. Add 0.8 g agarose to 100 ml 1X TAE and allow agarose to
melt by heating the solution (microwave oven/ heating
mantle)
3. Gently stir the solution intermittently till a transparent gel
like mixture is obtained
4. Do not allow to overheat and bubble formation
5. Allow to cool to warm and add 3 µl Ethidium Bromide with
the help of a pipette (Stock concentration - 10 mg/ml;
Working concentration - 1 μg/ml.)
6. Pour gel on gel casting tray (with suitable comb and
sealed)
7. Allow to solidify then gently remove the comb and
seals.
8. Place the gel on electrophoresis unit ‘tank’ along with
casting tray.
9. Mix loading dye with DNA as (5 µl dye + 2 µl DNA).
10. Load DNA samples (mixed with loading lye) over the
well formed by comb

*EtBr: Highly carcinogenic/ mutagenic – any

contact with skin, eyes or direct inhalation must be
avoided.
11. Run the gel by attaching cod to the power pack for 20
min
12. Gel documentation by using gel doc system
Results:
 During agarose gel electrophoresis of genomic DNA, it was found that the
resulting DNA fragments are visible as clearly defined bands.
 But some RNA contamination was found in sample 5 and 8 whereas, the
sample 2 and 3 showing the protein and degraded DNA.
 The DNA standard or ladder separated to a degree that allows for the
useful determination of the sizes of sample bands.
 Practical-5
PCR based RAPD analysis
Aim: To study the PCR amplification
of DNA with particular RAPD
primers at recommended annealing
temperature
Basic Principle
1. Polymerase Chain reaction (PCR) is an in vitro method of
nucleic acid synthesis by which a particular segment of DNA
can be specifically amplified.
2. PCR technology had a significant impact in almost all areas of
molecular biology and modification of the basic procedures has
allowed developing numerous assays for detecting variation at
the nucleotide level.
3. Random amplified polymorphic DNA, approach had been
widely used in plant molecular biology. William et al. (1990)
have developed RAPD to detect genetic polymorphism in crop
plants.
Basic Principle
4. RAPD PCR is typically carried out using random
oligonucleotide (decamers) primers (10 nt length) that flank the
DNA fragment to be amplified.
5. These primers anneal to opposite strands of the target sequence
and are oriented so that DNA synthesis by the polymerase
proceeds across the region between the primers.
6. Amplification is achieved through repeated cycles of
denaturation, annealing and extension of the annealed primers
with thermostable DNA polymerase (Taq Polymerase).
7. As the extension products are complimentary to and capable of
binding primers, successive cycles of amplification double the
amount of target DNA synthesized in the previous cycle.
8. The resulting amplified product can be visualized by running
through agarose gel (1.2 – 1.5 %) with ethidium bromide
(Et.Br) stain.
Chemicals required:
1. RAPD random primers (10 nt)
2. dNTPs
3. Template DNA,
4. PCR buffer
5. Taq DNA polymerase,
6. Agarose gel eletrophoresis reagents (1X TAE, Ethidium Bromide, Gel
loadng dye etc.)
7. Ultra pure water.

Material required
Sample DNA to be analysed (20 – 40 ng/µl)
 Labwares required:

1. PCR tubes
2. Micro pipette
3. Tips
4. Mini cooler tray
5. Ice bucket
Apparatus required
PCR thermocycler Agarose gel electrophoresis unit with
power pack
Procedure
1. Label 0.2 ml PCR tubes for each genomic DNA and arrange the tubes in
the rack.
2. Add 2l of template DNA to the tubes already labeled and keep in the
PCR tube rack.
3. Prepare the following reaction cocktail in a 1.5 ml Eppendorf tube for
required number of reactions plus two reactions to compensate the
pipetting loss.

Components Concentration Quantity

Sterile H2O -  11.86 l
PCR buffer with MgCl2 10X 2.00 l
dNTP mix 10mM 1.60 l
Primers 10mM 1.50 l
Taq Polymerase 1 U/l 0.80 l
Template DNA 20 - 50ng /l 2.00 l
Total  - 20.00l
4. N.B. While preparing the PCR reactions, it is important to
keep the reactions in ice, add the components in the order as
indicated.
5. Add 18l of the reaction mixture to the PCR tube, which is
already loaded with 2l of template DNA making the final
volume to 20l
6. Mix by flicking the tube repeatedly with your finger and
then centrifuge for a few seconds to bring the contents to
the bottom of the tube (do not use the vortex mixer to mix
the enzyme mixture as this inactivate the enzyme).
7. Perform the PCR reaction in a thermocycler following the
programme given below:

Steps Temperature (0C) Duration (min) Cycles Activity

1 95 0C 10:00 min Denaturation
1
2 95 0C 00:15 sec Denaturation
3 52 0C 00:30sec  36 Annealing
4 72 0C 1:30 min Extension
5 72 0C 10:00 min 1 Final extension
6 4 0C Till further use 1 Storage
8. Load the tubes in PCR thermcycler machine and set PCR as following:
10. Take out samples after completion of cycles and resolve in
agarose gel.
A. Load the RAPD products (10l) of individual reactions for
electrophoresis in 1.5% agarose gel prepared with 1X TAE buffer
(having ethidium bromide)
B. Run in 1X TAE buffer at 80 volt for 1 hours. Standard DNA molecular
weight marker must be added to one of the wells in the gel.
 After completion of the PCR, perform agarose gel
Observations: electrophoresis and compare the RAPD patterns of the
four different samples genomic DNA

Results
1. The RAPD pattern i.e. number and
size of the amplified PCR product
varies among different DNA samples
taken for study.
2. This happens due to the variation in
their genomic DNA sequences and
annealing sites of the random primer.
3. By doing this experiment one can
study the RAPD fingerprints for
differentiating individuals at genus
and species level. Lane 1: 100 bp DNA Ladder
4. The RAPD patterns of wheat and Lane 2: RAPD Pattern of wheat-1 genomic DNA
Lane 3: RAPD Pattern of wheat-2 genomic DNA
cotton show significant differences
Lane 4: RAPD Pattern of cotton-1 genomic DNA
but the RAPD patterns within the Lane 5: RAPD Pattern of cotton-2 genomic DNA
species of same genus i.e. wheat-1,
wheat-2 show less differences.