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LAB MANUAL ON
MOLECULAR BIOLOGY
LAB MANUAL ON
MOLECULAR BIOLOGY
Ruhi Dixit
SATYNDRA K. YADAV
Kartikay Bisen
CHETAN KESWANI
Ashwani Kumar
SURYA P. SINGH
Ashim Borah
Chetan Keswani
LAB MANUAL ON
MOLECULAR BIOLOGY

© SATYNDRAKESWANI
© CHETAN K. YADAV

First
First Edition
Edition:: 2016
2013

Price : Rs. 150/-

ISBN : 978-81-909182-7-5

Published by :
Media Associates
J-281/86, Kartar Nagar, Delhi-110053

Printed by :
B.K. Offset
Naveen Shahadara, Delhi-110032
This book is dedicated to
Mahamana Pandit Madan Mohan Malviya
Founder Banaras Hindu University
7
CONTENT
1. General Laboratory Procedures, Equipment Use and

Safety Considerations.....................................................9
2. Isolation of Genomic DNA from Bacterial Culture......22
3. Isolation of Plasmids from E. coli by Alkaline Lysis....26
4. Isolation of Genomic DNA from Fungal Cells.............32
5. Isolation of Genomic DNA from Plant Tissue by
CTAB Method...............................................................35
6. Isolation of Genomic DNA from Animal Tissues
by Phenol- Chloroform Method....................................40
7. Measurement of Quantity and Purity of
Genomic DNA..............................................................44
8. Agarose Gel Electrophoresis of Isolated
Genomic DNA..............................................................47
9. Polymerase Chain Reaction..........................................51
10. Isolation of Total RNA from Bacterial Cells................61
11. Total RNA Isolation with TRIZOL Reagent.................67
12. Extraction of Total Plant RNA......................................71
13. Total Protein Extraction with TCA-Acetone and
2D-Gel Electrophoresis.................................................81
14. Protein Quantification...................................................86
15. Western Blotting.........................................................103
• Commonly Used Biochemical Data................................108
General Laboratory Procedures, Equipment Use and Safety Considerations 9
1.
General Laboratory Procedures,
Equipment Use and Safety
Considerations
I. Safety Procedures
A. Chemicals
A number of chemicals used in any molecular biology labora-
tory are hazardous. All manufacturers of hazardous materials are
required by law to supply the user with pertinent information on
any hazards associated with their chemicals. This information is
supplied in the form of Material Safety Data Sheets or MSDS.
This information contains the chemical name, CAS number and
health hazard data, including first aid treatment, physical data,
fire and explosion hazard data, reactivity data, spill or leak pro-
cedures, and any special precautions needed when handling the
chemical. A file containing MSDS information on the hazard-
ous substances should be kept in the lab. In addition, MSDS
information can be accessed on internet. You are strongly urged
to make use of this information prior to using a new chemical
and certainly in the case of any accidental exposure or spill. The
instructor/lab manager must be notified immediately in the case
of an accident involving any potentially hazardous reagents. The
following chemicals are particularly noteworthy:
• Ethidium bromide - carcinogen
• Acrylamide - potential neurotoxin
• Phenol - can cause severe burns
10 Lab Manual on Molicular Biology
These chemicals are not harmful if used properly: always

wear gloves when using potentially hazardous chemicals and
never mouth-pipette them. If you accidentally splash any of these
chemicals on your skin, immediately rinse the area thoroughly
with water and inform the instructor. Discard the waste in ap-
propriate containers.
B. Ultraviolet Light
Exposure to ultraviolet light can cause acute eye irritation.
Since the retina cannot detect UV light, you can have serious
eye damage and not realize it until 30 min to 24 hours after ex-
posure. Therefore, always wear appropriate eye protection when
using UV lamps.
C. Electricity
The voltages used for electrophoresis are sufficient to cause
electrocution. Cover the buffer reservoirs during electrophoresis.
Always turn off the power supply and unplug the leads before
removing a gel.
D. General Housekeeping
All common areas should be kept free of clutter and all dirty
dishes, electrophoresis equipment, etc should be dealt with ap-
propriately. Since you have only a limited amount of space to
call your own, it is to your advantage to keep your own area
clean. Since you will use common facilities, all solutions and eve-
rything stored in an incubator, refrigerator, etc. must be labelled.
In order to limit confusion, each person should use his initials
or other unique designation for labelling plates, etc. Unlabelled
material found in the refrigerators, incubators, or freezers may be
destroyed. Always mark the backs of the plates with your initials,
the date, and relevant experimental data, e.g. strain numbers.
II. Preparation of Solutions

A. Calculation of Molar, % and “X” Solutions.
1. Molar solutions
A molar solution is one in which 1 litre of solution contains

the number of grams equal to its molecular weight, e.g. to make
up 100 ml of a 5M NaCI solution= 58.456 (MW of NaCI) gm/
mol x 5 moles/litre x 0.1 litre = 29.29 gm in 100 ml of solution
2. Percent solutions
Percentage (w/v) = weight (g) in 100 ml of solution; Percent-
age (v/v) = volume (ml) in 100 ml of solution, e.g. To make a
0.7% solution of agarose in TBE buffer, weight 0.7 of agarose
and bring up volume to 100 ml with TBE buffer.
3. “X” Solutions
Many enzyme buffers are prepared as concentrated solutions,
e.g. 5X or 10X (five or ten times the concentration of the work-
ing solution) and are then diluted such that the final concentra-
tion of the buffer in the reaction is 1X, e.g. To set up a restriction
digestion in 25μI, one would add 2.5μI of a 10X buffer, the other
reaction components, and water to a final volume of 25 μI.
B. P
reparation of Working Solutions from
Concentrated Stock Solutions
Many buffers in molecular biology require the same com-
ponents but often in varying concentrations. To avoid having
to make every buffer from scratch, it is useful to prepare several
concentrated stock solutions and dilute as needed. e.g. To make
100 ml of TE buffer (10mM Tris, 1 mM EDTA), combine 1 ml
of a 1 M Tris solution and 0.2 ml of 0.5 M EDTA and 98.8 ml
sterile water. The following is useful for calculating amounts of
stock solution needed: CixVi = CfxVf, where
Ci = initial concentration of stock solution; Vi= initial vol,
or amount of stock solution needed Cf = final concentration of
desired solution; Vf = final vol, or volume of desired solution.
C. Steps in Solution Preparation
1. Refer to a laboratory reference manual for any specific
instructions on preparation of the particular solution and
the bottle label for any specific precautions in handling the
chemical. Weigh out the desired amount of chemical(s).

Use an analytical balance if the amount is less than 0.1 g.
Place chemical(s) into appropriate size beaker with a stir
bar. Add less than the required amount of water. Prepare
all solutions with double distilled water. When the chem-
ical is dissolved, transfer to a graduated cylinder and add
the required amount of distilled water to achieve the final
volume in preparing solutions containing agar or agarose,
weigh. Weigh the agar or agarose directly into the final
vessel. If the solution needs to be at a specific pH, check
with calibrated pH meter. Autoclave at 121 0C for 20
min. Some solutions cannot be autoclaved, for example,
SDS. These should be filter sterilized through a 0.22μm
or 0.45μm filter. Media for bacterial cultures must be au-
toclaved the same day it is prepared, preferably within
an hour or two. Store at room temperature and check
for contamination prior to use by holding the bottle at
eye level and gently swirling it. Solid media for bacterial
plates can be prepared in advance, autoclaved, and stored
in a bottle. When needed, the agar can be melted in a
microwave, any additional components, e.g. antibiotics,
can be added and the plates can be poured.
2. Concentrated solutions, e.g. 1M Tris-HCI pH=8.0, 5M
NaCI, can be used to make working stocks by adding
autoclaved double-distilled water in a sterile vessel to the
appropriate amount of the concentrated solution.
D. Glassware and Plastic Ware

Glass and plastic ware used for molecular biology must be
scrupulously clean. Dirty test tubes, bacterial contamination and
traces of detergent can inhibit reactions or degrade nucleic acid.
Glassware should be rinsed with distilled water and autoclaved or
baked at 1500C for 1 hour. For experiments with RNA, glassware
and solutions are treated with diethyl-pyrocarbonate to inhibit
RNases which is resistant to autoclaving. Plastic ware such as pi-
pettes and culture tubes are often supplied sterile. Tubes made
of polypropylene are turbid and are resistant to many chemicals,

like phenol and chloroform; polycarbonate or polystyrene tubes
are clear and not resistant to many chemicals. Make sure that the
tubes you are using are resistant to the chemicals used in your
experiment. Micropipette tips and microfuge tubes should be
autoclaved before use.
III. Disposal of Buffers and Chemicals

1. Any contaminated, solidified agar or agarose should be
discarded in the trash, not in the sink, and the bottles
rinsed well.
2. Any media that becomes contaminated should be
promptly autoclaved before discarding it. Petri dishes and
other biological waste should be discarded in biohazard
containers which will be autoclaved prior to disposal.
3. Organic reagents, e.g. phenol, should be used in a fume
hood and all organic waste should be disposed of in a
labelled container, not in the trash or the sink.
4. Ethidium bromide is a mutagenic substance that should
be heat treated before disposal and should be handled
only with gloves. Ethidium bromide should be disposed
of in a labelled container.
5. Dirty glassware should be rinsed, all traces of agar or other
substance that will not come clean in a dishwasher should
be removed, all labels should be removed and the glass-
ware should be placed in the dirty dish bin. Bottle caps,
stir bars and spatulas should not be placed in the bins but
should be washed with hot soapy water, rinsed well with
hot water, and rinsed three times with distilled water.
IV. Equipment
General Comments
It is to everyone’s advantage to keep the equipment’s in good
working condition. As a rule of thumb, don’t use anything un-
less you have been instructed in the proper use. This is true not
only for equipment in the lab but also departmental equipment.
Report any malfunction immediately. Rinse out all centrifuge ro-
tors after use and in particular if anything spills. Please do not
waste supplies - use only what you need. If the supply is running
low, please notify either the instructor/lab manager before the
supply is completely exhausted. Occasionally, it is necessary to
borrow a reagent or a piece of equipment from another lab. Ex-
cept in an emergency, notify the instructor.
A. Micropipettes
Most of the experiments conducted in the laboratory will de-
pend on your ability to accurately measure volumes of solutions
using micropipettes. The accuracy of your pipetting can only be
as accurate as your pipette and several steps should be taken to
insure that your pipettes are accurate and are maintained in good
working order. If they need to be recalibrated, do so. Since the
pipettes will use different pipette tips, make sure that the pipette
tip you are using is designed for your pipette.
B. Using a pH Meter
Biological functions are very sensitive to changes in pH and
hence, buffers are used to stabilize the pH. A pH meter is an
instrument that measures the potential difference between a ref-
erence electrode and a glass electrode, often combined into one
combination electrode. The reference electrode is often AgCl2.
An accurate pH reading depends on standardization, the degree
of static charge, and the temperature of the solution.
C. Autoclave Operating Procedures

Place all material to be autoclaved in an autoclavable tray. All
items should have indicator tape. Make sure lids on all the bot-
tles are loose. Do not crowd large number of items in tray such
that one must allow sufficient air/steam circulation to reach the
appropriate temperature.
1. Make sure chamber pressure is zero before opening the

door.
2. Place items to be autoclaved in the autoclave and close
the door. Some autoclaves require that you also lock the
door after it’s closed.
3. Set time - typically 20 minutes(in addition to the time
taken to reach 121 0C).
4. Temperature should be set at 1210C already, but double-
check and change if necessary.
5. Start the cycle. In some autoclaves, the cycle starts auto-
matically at step 5,while in others, turn to “sterilize”.
6. At the end of the cycle, check that the chamber pressure
is zero and the temperature is <1000C.
7. Open the door and remove contents wearing gloves and
immediately tighten all the loose caps of the bottles.
Proper Use of the Microscope

1. When moving your microscope, always carry it with both
hands. Grasp the arm with one hand and place the other
hand under the base for support.
2. Turn the revolving nosepiece so that the lowest power ob-
jective lens is in position.
3. Place the microscope slide on the stage and fasten it with
the stage clips. You can push down on the back end of the
stage clip to open it.
4. Using the coarse adjustment, lower the objective lens
down as far as it will go without touching the slide. Note:
Look at the slide and lens from the side when doing this.
5. Look through the eyepiece and adjust the illuminator (or
mirror) and diaphragm for the greatest amount of light.
6. Slowly turn the coarse adjustment so that the objective
lens goes up (away from the slide). Continue until the
image comes into focus. Use the fine adjustment, if avail-
able, for fine focusing.
7. Move the microscope slide around so that the image is in
the centre of the field of view and re-adjust the mirror,

illuminator or diaphragm for the clearest image.
8. You should be able to change to the next objective lenses
with only slight focusing adjustment. Use the fine adjust-
ment, if available. If you cannot focus on your specimen,
repeat steps 4 through 7 with the higher power objective
lens in place. Do not allow the lens to touch the slide.
9. The proper way to use a monocular microscope is to look
through the eyepiece with one eye and keep the other eye
open (this helps avoid eye strain). If you have to close one
eye when looking into the microscope, it’s ok. Remem-
ber, everything is upside down and backwards. When you
move the slide to the right, the image goes to the left!
10. Do not touch the glass part of the lenses with your fin-
gers. Use only special lens paper to clean the lenses.
11. When finished, raise the tube, click the low power lens
into position and remove the slide.
Working with DNA

A. Storage
The following properties of reagents and conditions are
important considerations in processing and storing DNA and
RNA. Heavy metals promote phosphodiester breakage. EDTA is
an excellent heavy metal chelator. Free radicals are formed from
chemical breakdown and radiation and they cause phosphodi-
ester breakage. UV light at 260 nm causes a variety of lesions,
including thymine dimers and cross-linking. Biological activity
is rapidly lost. 320 nm irradiation can also cause cross-linking,
but less efficiently. Ethidium bromide causes photo oxidation
of DNA with visible light and molecular oxygen. Oxidation
products can cause phosphodiester breakage. If no heavy metal
is present, ethanol does not damage DNA. Nucleases are found
on human skin; therefore, avoid direct or indirect contact be-
tween nucleic acids and fingers. Most DNases are not very stable;
however, many RNases are very stable and can adsorb to glass
or plastic and remain active. 50C is one of the best and simplest
conditions for storing DNA. –200C causes extensive single and

double strand breaks. –700C is probably excellent for long-term
storage. For long-term storage of DNA, it is best to store in high
salt (>1M) in the presence of high EDTA (>10mM) at pH 8.5.
Storage of DNA in buoyant CsCl2 with ethidium bromide in the
dark at 50C is excellent. There is about one phosphodiester break
per 200 kb of DNA per year.
Sterile Techniques
1. All media, including plates, liquid media and top agar
must be autoclaved immediately after it is prepared. It is
best to prepare media in several small bottles, only open-
ing one at a time. Check the bottle for contamination
before you use it by gently swirling it and looking for
cloudy material in the centre. Always grow up a small
amount of broth alone when growing cells overnight. A
small amount of contamination is not always evident un-
til the media is incubated at 370C.
2. Use a flame on inoculating loops and on the lips of media
bottles before and after pipetting from them. Always use
a fresh, sterile pipette or pipette tip when pipetting cul-
ture media, and never go back into a media bottle or cell
culture with a used pipette.
3. To prevent wide-scale, untraceable contamination, each
person should have his own stock of liquid culture media,
agar plates, 100% glycerol, glycerol stocks of cells, etc.,
don’t share.
4. Overnight cultures should be grown only from a single
colony on a fresh plate or from a previously-tested glyc-
erol stock that was grown from a single colony. To pre-
pare an overnight culture from a glycerol stock, take an
individually wrapped 1 ml pipette and a culture tube of
media to the –800C freezer. Quickly remove the cap from
the freezer vial containing the glycerol stock, scrap a small
amount of ice from the surface of the culture, replace the
cap on the freezer vial, and place the pipette into the cul-
ture tube. Sufficient numbers of bacteria are present in

the ice in order for the culture to grow to saturation in 16
hours. Never let the glycerol stock thaw.
Working with E. coli
1. Small Scale Cultures
Experiments using E. coli cells should always be done on fresh
cultures, either from a freshly streaked plate or from a glycerol
stock. To grow a small scale E. coli culture, prepare 3-5 ml of LB
(or appropriate broth - include antibiotic if the culture contains
a plasmid) in two sterile 50 ml tubes. (Note: smaller tubes can be
used but the culture will not be appropriately aerated and hence
will not grow well and is not recommended). Inoculate one tube
with a single colony from a fresh plate or a scraping from a glyc-
erol stock. The second tube is used as a broth control. Incubate
both tubes at 370C, shaking overnight. Inspect the tubes next
morning. The control broth should be clear and the inoculated
culture should be turbid. Make a note of any debris found in the
tubes and only incubate longer if the culture is not dense. Always
use immediately and do not allow cells to overgrow. For some ap-
plications, cells can be stored at 40C for short periods prior to use.
2. Permanent Storage
For every culture used, in particular, for newly constructed
strains or for cells containing plasmids, a permanent glycerol
stock must be prepared as soon as the construct has been con-
firmed and this stock must be placed in the laboratory stock col-
lection with the appropriate documentation and location infor-
mation. This procedure pertains not only to E. coli but to any
organism for which a deep freeze stock can be prepared. Also,
all plasmid constructs, including construction intermediates,
must be maintained in cells, not as naked DNA stocks. For each
construct, at least 2 stocks should be made. To prepare a glyc-
erol stock for E. coli cells, combine 1.4 ml of a freshly grown
overnight culture with 0.6 ml of sterile 50% glycerol. Mix well.
Transfer to two freezer vials labelled with the strain name, the
date and your initials. Immediately place into a dry ice/ethanol

bath or into a box in the -800C freezer.
Instructions for maintaining lab book

1. A notebook should be kept for laboratory experiments.
The notebook should be written in ink, and each page
signed and dated. Mistakes are not to be erased but
should be marked out with a single line. Try to keep your
notebook with the idea that someone else must be able
to read and understand what you have done. The note-
book should always be up-to-date and can be collected
any time.
1. INDEX: An index containing the title of each experiment
and the page number should be included at the beginning
of the notebook.
2. What should be included in the notebook? Essentially
everything you do in the laboratory should be in your
notebook. Start each new experiment on a new page. The
top of the page should contain the title of the experiment,
the date and the page number. The page number is im-
portant for indexing, referring to previous experiments,
and for labelling materials used in a given experiment. If
an experiment spans more than one page, note the page
on which the experiment continues if it’s not on the next
page. Each experiment should include the following:
A. Title/Purpose/Aim: Every experiment should have a title
and it should be descriptive. When starting a new project,
it is a good idea to introduce the overall strategy prior to
beginning the first experiment. This serves two purposes.
First, it forces you to think about what you are doing
and why sometimes things look differently when written
down than they do in your head. Secondly, ideas can be
patented, and a thorough description of your hypothesis
and experimental strategy with appropriate documenta-
tion can be helpful for any future intellectual property
issues.
B. Background information: This section should include

any information that is pertinent to the execution of the
experiment or to the interpretation of the results. For ex-
ample, if it is a repeat experiment, state what will be done
differently to get the experiment to work? If it’s a clon-
ing experiment, include what the strategy is and how the
recombinants will be screened. A simple drawing of the
plasmid map can be helpful. This is not like the introduc-
tion to a paper. Include anything that will be helpful in
carrying out the experiment and deciphering the experi-
ment at a later date. For the most part, notebooks are not
written for today but for the future.
C. Materials: This section should include the key materials,
i.e., solutions or equipment that will be needed. It is not
necessary to include every piece of lab equipment required,
i.e. vortex, micropipette, etc., but you should include any
specialized equipment and the manufacturer, i.e., a phos-
phoimager or real-time PCR instrument. Composition
of all buffers should be included unless they are standard
or are referenced. Pre-packaged kits should be identified
as to the name of the kit, the vendor, and the catalogue
number. Biological samples should be identified by ge-
nus and species, strain number, tissue type, and/or geno-
type with the source of the material identified. Enzymes
should be identified by name, vendor, and concentration.
DNA samples should be identified as (a) Type of DNA,
i.e., chromosomal, plasmid, etc., (b) Purity (miniprep, gel
purified, PCR product) (c) Concentration, if known, and
(d) Source, (include prior experiment number if the DNA
was isolated in a previous experiment). Include all calcu-
lations made in preparing solutions. The sequence of all
oligonucleotides must be included or referenced. Agarose
gels should be identified by percentage and buffer used. If
any of these materials were used in previous experiments,
include only the reference to that earlier experiment, do
not repeat the information again.
D. Procedure/ Method: Write down exactly what you are

going to do before you do it and make sure you under-
stand each step before you do it. It is also a good laborato-
ry practice to have a separate notebook containing meth-
ods that you use on a regular basis. If an experiment is a
repeat of an earlier experiment, you do not have to write
down each step but refer to the earlier experiment by page
or experiment number. Flow charts are sometimes help-
ful for experiments that have many parts. Tables are also
useful if an experiment includes a set of reactions with
multiple variables. It is good practice to check off steps
as they are completed or reagents as they are added to
prevent you from losing you place or for forgetting to add
something. All procedures should be referenced.
E. Results: This section should include all raw data, includ-
ing gel photographs, printouts, colony counts, autoradio-
graph, etc. All lanes on gel photographs must be labelled
and always identify the source and the amount of any
standards. This section should also include your analysed
data, for example, transformation efficiencies, calcula-
tions of specific activities or enzyme activities.
F. Conclusions/Summary: This is one of the most impor-
tant sections. You should summarize all of your results,
even if they were stated elsewhere and state any conclu-
sions you can make. If the experiment didn’t work, what
went wrong and what will you do the next time to try to
trouble shoot?
2.
Isolation of Genomic DNA from
Bacterial Culture
Principle
Isolation of nucleic acids from your sample will be accomplished
in two steps. Firstly, direct extraction of nucleic acids from the
sample by bead beating and finally. Purification of nucleic acids
by phenol/chloroform cleanup method is followed by precipita-
tion in isopropanol.
Materials
1. Extraction buffer (pH 8.0): 50 mM NaCl
2. Tris-HCl, 50 mM, pH 7.6
3. EDTA 50mM
4. SDS 5%
5. Phenol (pH 8.0)
6. Chloroform: isoamyl alcohol : 24:1
7. Chloroform
8. Isopropanol 100%
9. Ethanol 70%
10. Microcentrifuge tubes
Method
A. Nucleic acid extraction
1. Grow an appropriate volume of bacterial culture to de-
sired OD. Isolating DNA from overgrown cells will result
Isolation of Genomic DNA from Bacterial Culture 23
in low yield; therefore, the culture should be in the log

phase to facilitate the most efficient extraction.
2. Centrifuge the bacterial suspension for 5 min at 4500
rpm to pellet the bacteria. Discard the supernatant.
3. Resuspend the pellet in 1 ml of extraction buffer by pipet-
ting up-and-down repeatedly. Do not vortex, as this will
cause considerable foaming and difficulty in transferring
the appropriate volume in subsequent steps. Transfer the
suspension to a sterile 2-ml microcentrifuge tube (with
locking lid) containing 0.4-0.5 ml of glass beads (0.10-
0.11 mm diameter).
4. Shake with Fast-Prep (tissue lyser) instrument for 15 sec.
at 4.0ms–1. Note: this is a different setting than for the soil
DNA extraction. A bead-beater rapidly shakes microcen-
trifuge tubes that contain sample and glass beads to lyse
cells and release DNA.
5. Centrifuge for 3 minutes at 14,000 rpm.
B. Nucleic acid purification

1. Add 300μl of both phenol and chloroform/ isoamyl al-
cohol. Vortex until an emulsion forms and the solution
appears milky (5-10 sec.). Centrifuge for 3min at 14,000
rpm or until phases are well separated. The aqueous phase
containing the DNA will be the upper phase. With a ster-
ile pipette tip, transfer the aqueous phase to a new 2 ml
tube.
2. Extract with 500μl of chloroform (not chloroform/ isoa-
myl alcohol) as above. Centrifuge as above, then transfer
aqueous phase to a new 1.5 ml tube.
C. Nucleic acid precipitation

1. Determine the volume of your extract. Add exactly 0.1
volumes of 3 M sodium acetate solution and 0.7 volumes
of isopropanol. Mix well by inverting the tube several
times. Do not vortex.
2. Precipitate the DNA by centrifugation at 14,000 rpm for

30 min in the refrigerated centrifuge (100C, tube hinges
pointing up).
3. Carefully discard the supernatant by aspirating the iso-
propanol. Isopropanol precipitated pellets may detach
from the side of the tube, so be careful not to loosen and/
or dislodge the pellet.
4. Wash the pellet by adding 0.5 ml ice cold 70% ethanol
and inverting the tube gently. Be sure that the ethanol
contacts all surfaces inside the tube. Re-pellet the DNA
again with centrifugation for 5 min. Optimization of this
protocol has shown that centrifugation at cool tempera-
tures (10-150C) will result in better pellet formation and
stability. Thus, the pellets are larger (containing more
DNA) and will stick to the sides of the tube, which makes
aspirating the alcohol easier. Remove ethanol as above be-
ing careful not to aspirate the pellet. Allow pellet to dry
for 2-5 minutes.
D. Nucleic acid resuspension and final cleanup

Resuspend the pellet by adding exactly 50μl of DNase/
RNase-free water and mixing by flicking the tube with your fin-
ger until the pellet dissolves.
Precautions
1. Wear gloves throughout the experiment.
2. Do not cross-contaminate your samples or the solutions.
Be aware of your pipette tip.
3. Work clean, either on fresh blue bench paper, in the hood,
or on a freshly ethanol treated bench top.
4. Perform all centrifugations with the hinge of the tube
pointing “up”.
5. Do not use a vortex at any point in this protocol unless
specified.
Isolation of Genomic DNA from Bacterial Culture 25
References
1. Sambrook J, Russell DW. 2001. Molecular cloning: A labo-
ratory manual 3rd edition, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, New York.
2. Baess, I. 1974. Isolation and puritication of deoxyribonucle-
ic acid from mycobacteria. Acta Pathologica Microbiologica
Scandinavia, Section B, 82, 780-784.
3. Troyer, D L. 1990. a rapid and simplified protocol for DNA
isolation from bacteria. Veterinary Research Communica-
tions, 14, 447-451.
3.
Isolation of Plasmids from E. coli by
Alkaline Lysis
Principle
Purification of plasmid DNA from Escherichia coli by alkaline
lysisis based on the differential denaturation of chromosomal and
plasmid DNA in order to separate the two. Bacteria are lysed with
a solution containing sodium dodecyl sulphate (SDS) and sodium
hydroxide. During this step, chromosomal as well as plasmid
DNA are denatured. Subsequent neutralization with potassium
acetate allows only the covalently closed plasmid DNA to
reanneal and to stay solubilised. Most of the chromosomal DNA
and proteins precipitate in a complex formed with potassium and
SDS, which is removed by centrifugation. The plasmid DNA
is concentrated from the supernatant by ethanol precipitation.
Using this procedure, 2–5μg of DNA can be obtained from a
1.5-mL culture of E. coli containing a pBR322 derived plasmids,
and three- to five-fold higher yields can be expected from pUC-
derived plasmids.
As with any plasmid isolation procedure, success in using the
alkaline lysis protocol is mainly dependent on the strain of E.
coli used. Strains that have a high endonuclease A activity, such
as HB101 or the JM100 series yield DNA that often necessitates
further purification with a phenol extraction and/or additional
precipitation. However, the alkaline lysis procedure seems to
be the most consistent plasmid purification protocol regardless
of the strain and it is also better suited for isolation of high-
Isolation of Plasmids from E. coli by Alkaline Lysis 27
molecular-weight (>10 kb) or low-copy-number plasmids than

is the boiling lysis method. Plasmid DNA isolated by alkaline
lysis is suitable for most analyses and cloning procedures without
further purification. However, if the isolated plasmid DNA is
to be sequenced, an additional purification step, such as phenol
extraction would improve the quality of plasmid DNA.
Materials
1. Growth of E. coli
1. Luria–Bertani (LB) medium: 5 gm/L yeast extract,
5 gm/L NaCl, 10 gm/L tryptone.
2. Appropriate antibiotics.
2. Plasmid Isolation
1. STE (sucrose/Tris/EDTA) solution: 8% (w/v) sucrose,
50mM Tris-HCl (pH 8.0), 50mM EDTA (pH 8.0).
Autoclave and store at 4°C.
2. GTE (glucose/tris/EDTA) solution: 50mM glucose,
25mM Tris-HCl (pH 8.0), 10mM EDTA (pH 8.0).
Autoclave and store at 4°C.
3. Alkaline–SDS solution: 0.2N NaOH, 1% (w/v) SDS.
Prepare fresh.
4. High-salt solution: 60 mL of 5 M potassium acetate,
11.5 mL glacial acetic acid, 28.5 mL DDW. The resulting
solution is 3 M acetate and 5 M potassium and has a pH
of about 4.8. Store at room temperature, do not autoclave.
5. TE buffer: 10mM Tris-HCl, pH 8.0; 0.1mM EDTA, pH
8.0, autoclave and store at room temperature.
6. Lysozyme:10mM Tris-HCl 20 mg/mL, pH 8.0.
7. RNase A: 10 mg/mL, DNase-free.
8. Ethanol 100% and 70%.
9. Phenol: chloroform (1:1).
Procedure
A. Growth of E. coli
1. Inoculate 3 mL of sterile LB medium containing the
appropriate antibiotic with a single bacterial colony.
2. Grow with shaking at 37°C overnight.

B. Plasmid Isolation
1. Centrifuge 1.5 mL of culture for 1 min. at maximum
speed in a microcentrifuge to pellet the bacteria. Decant
the supernatant.
2. Optional wash step: Resuspend the cell pellet in 0.5
mL STE solution. Centrifuge again and remove the
supernatant.
3. Resuspend the bacterial pellet in 100μL GTE Solution,
containing 2 mg/mL of lysozyme by vigorous vortexing.
4. Add 200μL of freshly prepared alkaline–SDS solution
and mix by inverting rapidly five times; do not vortex.
Place the tube on ice for 5 min.
5. Add 150μL of high-salt solution and vortex for 2 sec.
Place the tube on ice for 5 min.
6. Centrifuge at maximum speed for 5 min in a
microcentrifuge. Transfer the supernatant to a fresh tube.
7. Optional extraction with phenol: chloroform: Add
an equal volume of phenol: chloroform (1:1) and
mix by vortexing for 5 sec. Centrifuge for 1 min in a
microcentrifuge at maximum speed to achieve phase
separation. Transfer the top aqueousphase to a clean tube.
8. Precipitate DNA by adding 2 volumes of 100% ethanol.
Mix by vortexing and let stand for 2 min.
9. Centrifuge at maximum speed for 15 min in a
microcentrifuge at 4°C.
10. Carefully transfer the supernatant to a fresh tube and
add 1 mL of 70% ethanol and centrifuge at maximum
speed for 2 min in a microcentrifuge at 4°C. Remove the
supernatant as completely as possible and let the DNA
pellet air-dry for 10 min.
11. Dissolve the DNA pellet in 50μL of TE buffer containing
20μg/mL of RNaseA.
C. Plasmid Analysis
Analyse the DNA by agarose gel electrophoresis. It is
recommended that undigested supercoiled plasmid DNA be
analysed to verify the integrity of the DNA and to assess the
content of chromosomal DNA. In addition, analyse the DNA
by cleavage with restriction enzymes. Use 1μL of the plasmid
preparation in the case of high-copy-number plasmids (such as
pUC derivatives) and 3μL or more in the case of medium- or
low-copy-number plasmids. If the DNA is resistant to cleavage
with restriction enzymes, extract the isolated plasmid DNA with
phenol: chloroform and precipitate again with ethanol. The
concentration of the plasmid DNA can be roughly estimated by
comparing it to a plasmid of known concentration. Run 0.1μg
of the plasmid standard and several amounts(e.g., 1μL, 2μL, and
4μL) of the new plasmid prep. After ethidium bromide staining,
estimate the concentration of the new plasmid miniprep by
comparing the band intensity with those of the plasmid of known
concentration.
Nicked Uncut Cut

Linear
Covalently Linear
Boaded
Supercoiled
Circular
Sliagle
Stranded
Fig: Relative positions of different DNA forms of
a plasmid on a Tris-Acetate agarose gel.
D. Precautions
1. Insufficient removal of the growth medium and inadequate
washing of DNA pellets after ethanol precipitation are the
most common reasons for miniprep DNA being resistant
to cleavage with restriction enzymes.
2. Some bacterial strains shed cell wall components into
the medium that can inhibit the action of restriction
enzymes. This can be overcome by washing the cell pellet

in STE solution before lysing the bacteria.
3. Addition of lysozyme is not necessary for successful
plasmid isolation; however, adding lysozyme to the GTE
solution generally increases the DNA yield. However,
it may also increase the amount of contaminating
chromosomal DNA.
4. Alternatively, RNase can be added to the GTE solution
at a concentration of 100 μg/mL.The addition of RNase
A is optional, but recommended, because contaminating
RNA may interferes with the detection of DNA fragments
upon cleavage with restriction enzymes. It is important to
use DNase-free RNase, which is commercially available.
Alternatively, DNases can be inactivated by boiling the
RNase solution for 10 min.
5. Prolonged exposure of superhelical DNA to heat or alkali
results in irreversible denaturation. The resulting cyclic
coiled DNA migrates through agarose gels at about twice
the rate of superhelical DNA. It cannot be cleaved with
restriction enzymes, impairs sequencing of the DNA, and
stains poorly with ethidium bromide. Traces of this form
of DNA can often be seen in plasmid prepared by alkaline
or boiling lysis of bacteria.
6. Chromosomal DNA can be identified as a high-molecular-
weight band in an undigested plasmid preparation. Upon
cleavage with restriction enzymes, this band will disappear
and may result in a smear of DNA. In this case, omit the
lysozyme from the GTE solution, and, after addition of
the SDS–alkaline solution, mix by inverting rather than
by vortexing.
7. Do not dilute the nucleic acid below the minimal
concentration that allows the reliable quantification of
DNA. Optical density measurements of less than 0.05
are generally unreliable; ideally, samples should be diluted
so that they yield a minimum reading of 0.1.
References
1. Sambrook J, Russell DW. 2001. Molecular cloning: A
laboratory manual.3rd edition, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, New York.
2. Birnboim, H. C. and Doly, J. 1979. A rapid alkaline
extraction procedure for screening recombinant plasmid
DNA. Nucleic Acids Res. 7, 1513–1523.
3. Birnboim, H. C. 1983. A rapid alkaline extraction method
for the isolation of plasmid DNA. Methods Enzymol. 100,
243–255.
4.
Isolation of Genomic DNA from
Fungal Cells
Principle
Isolation of DNA from fungal mycelia and single cells, this pro-
tocol is widely used and has many variations. This method can
also be used to obtain DNA from fungal material including fruit-
ing bodies and cultures, dried herbarium specimens, frozen sam-
ples, and freeze-dried material.
Reagents
1. Extraction buffer (EB): 0.15M NaCl, 50nM Tris (pH
8.0), 50mM EDTA, 1% SDS
2. Phenol-Chloroform (PC)
3. Chloroform-Isoamyl alcohol 24:1 (CI)
4. TE-buffer: 10mM Tris Base, 1mM EDTA (pH 8.5)
5. 3M Na-acetate (pH 5.2)
6. RNAse 10 mg/ml
7. 95% and 70% EtOH
Tissue preparation
Cultured mycelium may be dried or fresh. In either case,
harvest by filtration onto filter paper with a funnel, wash with
DDW, remove agar inoculum block if present. Scrape off myce-
lium growing on solid media using a sterile scalpel. Fruiting body
Isolation of Genomic DNA from Fungal Cells 33
tissue may be dried or fresh. Scrape clean or cut away surface

tissues. Use approximately 20 mg from dried samples or 200 mg
from fresh material.
Procedure
1. Samples may be ground dry or in liquid nitrogen. A pinch
of sterile sand helps in better grinding.
2. Dried cultured mycelium and small, delicate dried fruit-
ing body fragments are best ground dry in an eppendorf
tube with blue pellet pestles. Grind dry, then add about
500ul hot EB, place in 650C water bath. Leave pestle in
sample and regrind occasionally over 15-60 min.
3. Large amounts of mycelium or larger or tougher fruiting
bodies are best ground in porcelain mortar with liquid
nitrogen. Grind to fine powder. Can use small amount of
powdered mycelium for minipreps in eppendorf tubes or
larger amounts for maxiprep in oakridge/corex tubes. For
maxiprep, add mycelium to oak ridge tube (polypropyl-
ene, not polycarbonate) and add 5-10 ml hot extraction
buffer. Place in 650C water bath for 15-60 min with oc-
casional mixing.
4. Add equal volume phenol-chloroform as extraction
buffer. Mix several times to form emulsion. (If necessary,
transfer to corex tube at this point). Spin for 10 min in
microfuge. Carefully remove aqueous, upper layer (con-
taining DNA) to a new tube.
5. Repeat step 4, but this time use chloroform-isoamyl alco-
hol instead phenol-chloroform.
6. Repeat step 5.
7. Add 0.1 volumes 3M Na-acetate and 1.8 volumes cold
95% EtOH. Mix gently. This can be done in polycarbon-
ate.
8. If visible precipitate forms, spin down immediately, 5-10
min. If no DNA is seen, put at –200C for 15 min and then
spin.Draw off EtOH. Add 70% EtOH (same volume as
95%) and mix gently for about 1 min. Draw off 70% or
spin 5 min first and (air) dry DNA pellet.
9. Resuspend in 500 / 50 µl (maxiprep / miniprep) TE
buffer.
10. Often, there are large amounts of RNA in the DNA solu-
tions. If desired, the RNA can be removed by a 50 min
room temperature digestion with 0.01 volumes 10mg/ml
RNAse, followed by two CI extractions and EtOH pre-
cipitation.
References
1. Sambrook J, Russell DW. 2001. Molecular cloning. A labo-
ratory manual, 3rd edition, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, New York.
2. Al-Samarrai, T. H. and Schmid, J. 2000. A simple method
for extraction of fungal genomic DNA. Letters in Applied
Microbiology, 30, 53–56.
3. Raeder, U. and Broda, P. 1985. Rapid preparation of DNA
from filamentous fungi. Letters in Applied Microbiology, 1,
17–20.
4. Blin, N. and Stafford, D. W. 1976. A general method for
isolation of high molecular weight DNA from eukaryotes.
Nucleic Acids Research, 3, 2303–2308.
Isolation of Genomic DNA from Plant Tissue by CTAB Method 35
5.
Isolation of Genomic DNA from Plant
Tissue by CTAB Method
Principle
The DNA extraction from plant tissue involves digestion of cell
wall, followed by disruption of cell membrane to release nuclear
DNA into the extraction buffer. This is normally achieved by ce-
tyltrimethyl ammonium bromide (CTAB), a cationic detergent,
which forms complexes with proteins. The released DNA should
be protected from endogenous nuclease. Since, DNase activity
has pH optima of 5.0 and depends upon Mg++ ions, a buffer sys-
tem having alkaline pH and with divalent cation chelator is used
to inactivate DNase. Hence, Tris-Cl buffer (pH 8.0), with EDTA
is used to inhibit DNase in initial steps of homogenization.
Chloroform causes surface denaturation of proteins and isoa-
myl alcohol reduces foaming and stabilizes the interface between
the aqueous phase and organic phase, where protein collects.
Then upper phase containing nucleic acid is separated and DNA
is precipitated by the addition of absolute ethanol and sodium
acetate. Sodium acetate reacts with phosphate group of DNA
which helps in precipitation of DNA and giving a thread-like
appearance.
Reagents
1. Autoclaved double distilled water.
2. Tris-Cl (1M, pH 8.0): Dissolve 12.1 gm of Tris base
(M.W. 121.1) in 70 ml of DDW. Adjust pH to 8.0 by
slowly adding 6N HCl and make up the volume of solu-

tion to 100 ml with DDW. Sterilize by autoclaving and
store at room temperature.
3. EDTA (0.5 M, pH 8.0): Add 18.6gm EDTA (M.W.
372.2) to 70 ml of DDW. Stir vigorously on a magnetic
stirrer. Adjust the pH to 8.0 with 1N NaOH. Adjust the
volume of the solution to 100 ml and sterilize by auto-
claving.
4. CTAB (10% w/v): Dissolve 10 gm of CTAB (M.W.
364.46) in 90 ml of DDW. Incubate and stir at 600 C
on a magnetic stirrer. Adjust the volume to 100 ml with
DDW and store at room temperature. Do not autoclave.
5. ß-mecaptoethanol.
6. Polyvinylpyrrolidone (PVP): 2% (w/v): Dissolve 2g of
PVP in 95ml of DDW and adjust the final volume to
100 ml with DDW.
7. TAE Buffer (50X): 24.2g Tri base, 5.71 ml of glacial ace-
tic acid and 10 ml of 0.5 M EDTA (pH 8.0) and make up
the volume 100 ml with DDW. Sterilize by autoclaving
and store at room temperature.
8. DNA Extraction Buffer:
Take the following components from the stock solu-
tion and make the desired final concentration by taking
volumes given in the table below.
Sl. No. Components Stock Volume Final conc.
conc. (ml)
1 Water – 5.2 ml –
2 Tris-Cl, pH 8.0 1M 10 100mM
3 NaCl 5M 30 1.5 M
4 EDTA 0.5 M 4 20 mM
5 CTAB 10 % 2 2%
6 ßmercaptoethanol – 2 2%
9. Sodium acetate (3 M, pH 8.0): Dissolve 24.6 g of sodium

acetate (M.W. 82.03) in 70 ml DDW. Adjust the pH to
5.0 with acetic acid and make final volume to 100 ml.
10. RNase A (10 mg/ml)
11. Phenol (M. W. 118.38)
12. Chloroform (M.W. 118.38)
13. Isoamyl alcohol (M.W. 88.15)
14. Glacial acetic acid (M.W. 60.05)
15. Absolute ethanol.
16. TAE: Prepare the solution with final concentration of 10
mM Tris-Cl( pH 8.0 )and 1mM EDTA (pH 8.0) by tak-
ing stock solution 2 and 3 and store at 40C.
Collection Of Plant Materials

Collect the young and tender leaves from the selected plant
species and keep them in a plastic bag on ice. Immediately trans-
port the samples to the laboratory and transfer the fresh sample
in a falcon tube and pour liquid nitrogen over the sample, let it
evaporate then store at- 200C.
Procedure
1. In a pre-chilled, sterile mortar and pestle grind 1gm of
frozen leaf sample in liquid nitrogen till fine powder is
obtained.
2. Add 5 ml of pre-warmed (600C) DNA extraction buffer
to mortar and pestle.
3. Pour the slurry in a sterilized 15 ml polypropylene cen-
trifuge tube.
4. Add 100 mg polyvinyl pyrrolidone (PVP) and invert the
tubes several times to mix thoroughly.
5. Incubate at 600C for 1 hour and cool to room temperature.
6. Add equal volume of chloroform: isoamyl alcohol (24:1)
and mix gently by inverting the tubes 20 to 25 times to
form an emulsion.
7. Centrifuge at 12,000 rpm for 20 minutes at room
temperature.
8. Transfer the top aqueous phase to a new 15 ml centrifuge

tube. A second chloroform: isoamyl alcohol (24:1)
extraction may be performed if the aqueous phase is
cloudy due to the presence of PVP.
9. Add 2 volumes of pre-chilled (-200C) 95% ethanol and
sodium acetate (final conc. 0.3 M).
10. Mix gently by inverting and keep at -200C for 30 minutes.
11. Spin at 3000 rpm for 5 minutes and then increase speed
to 10,000 rpm for an additional 5 minutes at room tem-
perature. This differential spinning step helps to keep
DNA at the bottom of the centrifuge tube.
12. Pour off the supernatant and wash the pellet twice
(centrifuge at 10,000 rpm for 5 minutes at room
temperature) with 2 ml of 75 % ethanol.
13. Decant the supernatant and the DNA pellet is kept at
room temperature for air drying until the whitish pellet
turns transparent (20 to 30 minutes).
14. Dissolve the dried DNA pellet in 200 to 300 µl of TAE
and store at 40C for further use.
RNase Treatment
1. Prepare the assay mixture by taking 100 µl DNA sample,
98 µl TAE and 2 µl RNaseA (10 mg/ml).
2. Incubate the above mixture at 37 0C for 1 hour.
3. Add 200 µl of phenol: chloroform (3:1).
4. Mix gently by inverting the tubes 20 times and keep at
room temperature for 2-3 minutes.
5. Centrifuge at 10,000 rpm for 5 minutes at 40C.
6. Take the aqueous phase and precipitate with 2 volumes
of 95% ethanol and sodium acetate (final conc. 0.3M).
7. Keep at -200C for 30 minutes.
9. Pour off the supernatant and wash the pellet twice (cen-
trifuge at 10,000 rpm for 5 minutes at room tempera-

ture) with 2 ml of 75% ethanol.
10. Decant the supernatant and the DNA pellet is kept at
room temperature for air drying until the whitish pellet
turns transparent (20 to 30 minutes).
11. Dissolve the DNA pellet in minimum volume (23-30μl)
of TAE and store at 40C for further use.
12. Measure the quantity and purity of isolated DNA by
spectrophotometer or observe DNA band on agarose gel.
Precautions
1. Care should be taken while separating the aqueous phase,
if it is cloudy the same step should be repeated twice.
2. Avoid samples and reagents defreezing and warming
unless the experiment procedure advice to do it.
3. To avoid mechanical shearing, DNA pellet should not be
agitated during dissolving in TAE.
4. Phenol, ß-mercaptoethanol, chloroform are toxic and
corrosive, avoid contact with skin and eyes, do not
breathe vapours.
References
1. Doyle, J.J. and Doyle, J.L. 1987. A rapid DNA isolation
procedure for small quantities of fresh leaf tissue.
Phytochemistry Bulletin. 19, 11-15.
2. Stewart, C.N. and Via L.E. 1993. A rapid CTAB DNA
isolation technique useful for RAPD fingerprinting and
other PCR applications. BioTechniques article Vol. 14(5),
748-749.
3. Sambrook, J, Russell DW. 2001. Molecular Cloning. A
laboratory manual. Third Edition. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, New York.
6.
Isolation of Genomic DNA from Animal
Tissues by Phenol- Chloroform Method
Principle
Intact genomic DNA is obtained by lysing the cells and inhibit-
ing DNase activity in cells. Since, DNase activity has pH optima
of 5.0 and depends upon Mg++ ions. A buffer system having al-
kaline pH and with divalent cation chelator is used to inactivate
DNase. Hence, Tris-Cl buffer (pH 8.0), with EDTA is used to
inhibit DNase in initial steps of homogenization. SDS is an ani-
onic detergent used for cell lysis to release its contents. RNase A
is used to digest RNA. Proteinase K is used to digest protein in
solution including enzyme RNase A.
To precipitate the proteins from the homogenate, phenol is
used along with chloroform, by means of which the solution is
separated into two distinct layers, upper aqueous phase contains
nucleic acid and lower phase contains the organic compound
and the denatured proteins remain in the interface. Chloroform
causes surface denaturation of proteins whereas; isoamyl alcohol
reduces foaming and stabilizes the interface between the aque-
ous phase and organic phase, where proteins collect. Then upper
phase containing nucleic acid is separated and DNA is precipi-
tated by the addition of absolute ethanol and sodium acetate.
Sodium acetate reacts with phosphoric acid of DNA which helps
in precipitation of DNA and forms a thread-like appearance.
Isolation of Genomic DNA from Animal Tissues by Phenol- Chloroform Method 41
Reagents
1. Tris-Cl (1M, pH 8.0): Dissolve 12.1gm of Tris base
(M.W. 121.1) in 70 ml of autoclaved double distilled wa-
ter. Adjust pH to 8.0 by slowly adding 6N HCl, finally
make up the volume up to 100 ml with sterile water. Ster-
ilize by autoclaving store at room temperature.
2. EDTA (0.5M, pH 8.0): Add 18.6gm of EDTA (M.W.
372.2) to 70 ml of autoclaved double distilled water. Stir
vigorously on a magnetic stirrer. Adjust the pH to 8.0
with 1N NaOH. Adjust the volume of the solution to
100 ml and sterilize by autoclaving. The EDTA will not
dissolve until the pH of the solution is adjusted to 8.0;
3. 10% Sodium Dodecyl Sulphate (SDS) (w/v): Dissolve
10gm of SDS in 90 ml of autoclaved double distilled wa-
ter. Heat to 680C and mix by swirling. Adjust the volume
to 100 ml with double distilled water. Store at room tem-
perature.
4. TAE- Buffer (50X): Take 24.2gm Tris base, 5.71 ml gla-
cial acetic acid and 10 ml of 0.5 M EDTA (pH 8.0) and
make up the volume 100 ml with DDW. Sterilize by au-
toclaving and store at room temperature.
5. Lysis buffer: Take the following components from the
stock solution and make the desired final concentration
by taking volumes given in the table below:
S. No. Components Stock conc. Volume ( ml ) Final conc.
1 Water – 14.8 –
2 Tris-Cl, pH 8.0 1M 0.2 0.01
3 EDTA 0.5M 4.0 0.1
4 SDS 10% 1.0 0.5%
6. Sodium acetate (3 M, pH 8.0): Dissolve 24.6gm of so-

dium acetate (M.W. 82.03) in 70 ml DDW. Adjust the
pH to 5.0 with acetic acid and volume to 100 ml.
7. Proteinase K (3 mg/ml)
8. RNase A (10mg/ml)
9. TE Buffer: Prepare the solution with final concentration
of 10 mM Tris-Cl (pH 8.0) and 1mM EDTA (pH 8.0) by
taking stock solution 2 and 3 and store at 40 C.
Procedure
1. Make 10% homogenate (0.5gm tissue + 0.4ml lysis buff-
er) with the help of a sterile mortar and pestle and trans-
fer to a 1.5 ml micro centrifuge tube.
2. Mix properly and stand for 5 minutes.
3. Add 10 µL of 10 mg/ ml RNaseA, vortex for 30 seconds
and incubates at 370 C for 1 hr.
4. Then add proteinase K (final conc. 100 µg/ ml), vortex
for 30 sec and incubate at 50 0 C for 1 hr in a preheated
water bath.
5. Add equal volume of phenol and mix properly by invert-
ing the tube gently.
7. Take the aqueous phase and add equal volume of PCI
(Phenol: Chloroform: Isoamyl alcohol: 25:24:1). Mix
gently by inverting.
9. Take the aqueous phase and add equal volume of chlo-
roform.
11. Take the aqueous phase and add 2 volumes of absolute
alcohol and sodium acetate. Mix gently by inverting and
keep at -200C for 30 minutes.
13. Discard the supernatant and wash the pellet with 500 µl
of 75% alcohol (centrifuge at 12,000 rpm for 5 minutes
at 40C).
Isolation of Genomic DNA from Animal Tissues by Phenol- Chloroform Method 43
14. Decant the supernatant and the DNA pellet is left at

room temperature for drying until the whitish pellet turn
to transparent (avoid over drying).
15. Dissolve the dried DNA pellet in 20 µl of TAE and store
at 40C for further use.
Precautions
1. Care should be taken, when separating the aqueous phase
in step 5, 7 & 9.
2. Balancing of centrifuge tubes is necessary before centrifu-
gation.
3. To avoid mechanical shearing DNA pellet should not be
agitated during dissolving in TE buffer.
References
1. Qi Wu et al. 1995. A simple, rapid method for isolation
of high quality genomic DNA from animal tissues. Nucleic
Acids Research, Vol. 23, No. 24 5087-5088.
2. Gustincich S et al. 1991. A fast method for high-quality
genomic DNA extraction from whole human blood.Biotech-
niqes, 11(3), 298-300.
3. Gilber JR and Vance JM. 2001. Isolation of genomic DNA
from mammalian cells. Current Protocol in Human Genetics.
7.
Measurement of Quantity and Purity of
Genomic DNA
A. Estimation of DNA by Nanodrop

Principle
It is often difficult to measure the concentration of high
molecular weight DNA by standard methods such as absorbance
at 260 nm because the DNA solution is non-homogeneous
and often highly viscous that it becomes almost impossible to
withdraw a large amount of sample for analysis. A solution with
value of 1(A260measurement) contains 50 µg of DNA/ml. Note
that estimates of purity of nucleic acids based on OD260/OD280
ratios of ~1.2.
Calculation
Observed OD x50 µg/ ml x Dilution factor = Amount of
DNA (µg/ml)
Precautions
1. Significant absorbance at 270 nm generally indicates
phenol contamination. An OD260: OD 270 ratio of 1.2
indicates a clean DNA sample. If your ratio is lower, bear
in mind that there be some phenol contamination and
as a result the DNA measurement may be too high.
2. Absorbance at 230 nm is due to the presence of
organic compounds and is generally not a concern.
Measurement of Quantity and Purity of Genomic DNA 45
Often DNA isolated with a commercial kit shows

a significant peak at 230 nm. Phage genomic DNA
samples (and in particular concentrated ones) are
very difficult to accurately quantify because the DNA
tends to aggregate. Heat these samples for 15 min
at ~55°C before measuring to help break up clumps. 
3. If you’re having trouble getting consistent readings from
the same tube, it is recommended to dilute the sample
down to 100-200 ng/μl range. Do not vortex since it may
fragment DNA sample. 
B. Estimation of DNA by Diphenylamine

(DPA) Method
Principle
This is a general reaction given by deoxypentoses. The
2-deoxyribose of DNA, in the presence of acid, is converted to
ω-hydroxilevulinic aldehyde, which reacts with diphenylamine
to form a blue coloured complex, which can be read at 595 nm.
Reagents
1. Standard DNA solution- Dissolve DNA (200µg/ml) in
1N perchloric acid/buffered saline.
2. Diphenylamine solution- Dissolve 1gm of diphenylamine
in 100 ml of glacial acetic acid and 2.5 ml of concentrated
H2SO4. This solution must be prepared freshly.
3. Buffered Saline- 0.5 mol/litre NaCl; 0.015 mol/litre so-
dium citrate, pH 7.
Procedure
1. Pipette out 0.0, 0.2, 0.4, 0.6, 0.8 and 1 ml of working
standard in to the series of labelled test tubes.
2. Pipette out 1 ml of the given sample in another test tube.
3. Make up the volume to 1 ml in all the test tubes. A tube

with 1 ml of distilled water serves as the blank.
4. Now add 2 ml of DPA reagent to all the test tubes
including the test tubes labelled ‘blank’ and ‘unknown’.
5. Mix the contents of the tubes by vortexing / shaking the
tubes and incubate on a boiling water bath for 10 min.
6. Then cool the contents and record the absorbance at 595
nm against blank.
7. Then plot the standard curve by taking concentration
of DNA along X-axis and absorbance at 595 nm along
Y-axis.
8. Then plot the standard curve and calculate the
concentration of DNA in the given sample (unknown).
Volume of Volume Concentration Volume Incubate Abs. at
standard of distilled of DNA (µg) of DPA in 595 nm
reagent
(200 µg/ml) water (ml) boiling
(ml)
DNA (ml) water
0.0 1.0 00 2 bath 0.00
0.2 0.8 40 2 for
0.4 0.6 80 2 10 min
0.6 0.4 120 2 &
0.8 0.2 160 2 Cool
1.0 0.0 200 2
Unknown 0.0 ? 2
1.0
Fig. Standard curve of DNA.
References
1. Ausubel FM. Brent R. Kingston RE. Moore DD. Seidman
JG. Smith JA. And K Struhl (eds) (1998) Current Protocols
in Molecular Biology. John Wiley & Sons.
2. Sambrook J. Fritsch E.F. and T. Maniatis (1989). Molecular
Cloning A Laboratory Manual. Second edition. Cold Spring
Harbor Laboratory Press.
Agarose Gel Electrophoresis of Isolated Genomic DNA 47
8.
Agarose Gel Electrophoresis of Isolated
Genomic DNA
Principle
When an electric field is applied across the gel, DNA which is
negatively charged at neutral pH migrates towards anode. The
rate of migration is determined by a number of parameters like
molecular size of the DNA, agarose concentration, conformation
of DNA, voltage applied, presence of intercalating dyes and
composition of electrophoresis buffer. Molecules of double
stranded DNA migrate through the gel matrices at a rate that is
inversely proportional to the log of the number of the base pairs.
Thus, larger molecules migrate slowly due to greater frictional
resistance than the smaller molecules.
Concentrations of agarose used for different size of DNA molecules.
Concentration. Of No. of base pairs
agarose(%)
0.5 700bp to 25 kb
0.8 500bp to 15 kb
1 250bp to 12 kb
1.2 150bp to 6 kb
1.5 80 p to 4 kb
The rate of electrophoretic mobility of DNA is given by:
Log µ = log µ0 – Kr i
Where,
µ= electrophoretic mobility of DNA
µ0= free electrophoretic mobility of DNA
Kr=retardation coefficient
i= concentration of gel (%)
Ethidium bromide is a fluorescent dye that intercalates DNA
double helix between adjacent base pairs. This intercalation
results in partial unwinding of the double helix. The DNA-
ethidium bromide complex strongly absorbs UV light at 300 nm
and band appears in orange region of visible spectrum.
Reagents
1. Ethanol (75%).
2. 50X TAE: Take the components i.e. 24.2 gm Tris base,
5.71 ml glacial acetic and to ml of 0.5 M EDTA (pH 8.0)
and make up the volume 100 ml with DDW. Sterilize by
autoclaving and store at room temperature.
3. 1X TAE: Take 10 ml of 50X TAE in 490 ml DDW.
4. Ethidium bromide (10 mg/ml): Dissolve 100 mg of EtBr
in 10 ml of DDW. Stir on a magnetic stirrer until the
dye has dissolved. Dispense into 500 µl aliquots and
wrap the container in aluminium foil and store at room
temperature. Final working concentration is 0.5 µg/ml.
5. Gel loading dye (6X): Take 0.25% Bromophenol blue,
0.25% Xylene cynol in 30% glycerol and store at 40C as
1 ml aliquots.
Procedure
1. Clean the gel tray and the comb with 75% alcohol.
2. Seal the open ends of the clean plate by the cellophane
tape and keep on a flat bench.
3. Take 25 ml of 1X TAE in a conical flask and add 0.25 gm
agarose (for 1% agarose gel).
4. Boil till the solution is transparent.
Agarose Gel Electrophoresis of Isolated Genomic DNA 49
5. Let the solution cool down to room temperature then add

5μl of ethidium bromide mix thoroughly.
6. Place the comb on the tray to get complete wells.
7. Pour the agaroge solution in the sealed tray and allow the
gel to solidify (25-30 min).
8. Remove the comb carefully after the gel has solidified;
transfer the gel to the electrophoresis tank containing 1X
TAE electrophoresis buffer.
9. Prepare the sample (DNA) with the gel loading dye and
buffer (gel loading dye 1.5 µl, 1X TAE 5.5 µl and isolated
sample DNA 2.0 µl) and carefully load the sample in the
wells of submerged gel electrophoresed at 80 volt and 100
mA current.
10. After 45 minutes visualize the gel in an UV- trans
illuminator.
(1) (2) (3)
(4) (5) (6)
Fig. Agarose Gel Electrophoresis.

Precautions
1. Same batch of electrophoresis buffer should be used in
both the electrophoresis tank and gel preparation.
2. Stock solution of ethidium bromide should be stored in
light protected containers at room temperature.
3. After pouring the agarose, the air bubbles should be
removed under the teeth of the comb.
4. Care should be taken at the time of loading, so that the
sample will not spill-out from the well.
References
1. Sambrook J. et al. (1989). Molecular Cloning A Laboratory
Manual. Second edition. Cold Spring Harbor Laboratory
Press.
2. Ausubel F.M. et al. (eds) (1998). Current Protocols in
Molecular Biology. John Wiley & Sons.
Polymerase Chain Reaction 51
9.
Polymerase Chain Reaction
Inrtoduction
As originally developed, the PCR process amplified short seg-
ments of approximately 100-500bps. A typical amplification
reaction includes the sample of target DNA, a thermostable
DNA polymerase, two oligonucleotide primers, deoxynucleo-
tide triphosphates (dNTPs), reaction buffer, magnesium and
optional additives. The components of the reaction are mixed
and the reaction is placed in a thermal cycler, which is an au-
tomated instrument that takes the reaction through a series of
different temperatures for varying amounts of time. This series of
temperature and time adjustments is referred to as one cycle of
amplification. Each PCR cycle theoretically doubles the amount
of targeted template sequence (amplicon) in the reaction. Ten
cycles theoretically multiply the amplicon by a factor of about
one thousand; 20 cycles, by a factor of more than a million in a
matter of hours.
Each cycle of PCR amplification consists of a number
of steps. Each step denatures the template producing two
oligonucleotide-primed single-stranded DNA templates, sets
up the polymerization reaction and synthesizes a copy of each
strand of the template being targeted. These steps should be
optimized for each template and primer pair combination. The
initial step in a cycle denatures the target DNA by heating it to
95°C or higher for 15 sec to 2 min. In the denaturation process,
the two intertwined strands of DNA separate from one another,
producing the necessary single-stranded DNA template for the

thermostable polymerase. The next step of a cycle reduces the
temperature to approximately 40-60°C. At this temperature, the
oligonucleotide primers can form stable associations (anneal) with
the separated target DNA strands and serve as primers for DNA
synthesis by a thermostable DNA polymerase. This step lasts
approximately 30-60 seconds. Finally, the synthesis of new DNA
begins when the reaction temperature is raised to the optimum
for the thermostable DNA polymerase. For most thermostable
DNA polymerases this temperature is approximately 74°C.
Target Region
Unamplified DNA
Cycle 1
Denature and anneal
primers
Extended primers
Cycle 2
Denature and anneal

primers
Extended primers
Cycle 3
Denature and anneal

primers
Extended primers
=Short ‘target’
product
=Short ‘target’
Cycle 4-30 product
Application of Short ‘target’ product
Fig: PCR Amplification

Extension of the primer by the thermostable polymerase lasts

approximately 1-2 min. This step completes one cycle, and the
next cycle begins with a return to 95°C for denaturation. After
20-40 cycles, the amplified nucleic acid may then be analyzed
for size, quantity, sequence, etc., or used in further experimental
procedures (e.g., cloning).
PCR Optimization
Magnesium Concentration
Magnesium concentration is a crucial factor affecting the
performance of Taq DNA polymerase. Reaction components,
including template DNA, chelating agents present in the sam-
ple (e.g., EDTA or citrate), dNTPs and proteins, can affect the
amount of free magnesium. In the absence of adequate free mag-
nesium, Taq DNA polymerase is inactive. Conversely, excess free
magnesium reduces enzyme fidelity and may increase the level
of nonspecific amplification. For these reasons, it is important
to empirically determine the optimal MgCl2 concentration for
each reaction. To do so, prepare a reaction series containing 1.0-
3.0mM Mg2+ in 0.5mM increments by adding 2, 3, 4, 5 or 6μl
of a 25mM MgCl2 stock to 50μl reactions.
First, completely thaw the magnesium solution prior to use;
second, vortex the magnesium solution for several seconds prior
to pipetting. Magnesium chloride solutions form a concentra-
tion gradient when frozen and vortexing is required to obtain
a uniform solution. These two steps, though seemingly simple,
eliminate the source of many failed experiments.
Enzyme Choice
The choice of the correct enzyme(s) to use in the PCR reac-
tion is determined by several factors. Taq DNA polymerase, the
first enzyme used for PCR, is still the most popular. This polymer-
ase possesses relatively high processivity and is the least expensive
choice. Taq DNA polymerase generates PCR products with sin-
gle deoxyadenosine overhangs on the 3´-ends. These overhangs
allow easy cloning into “T”-vectors (pGEM-T) which possess

“T” overhangs complementary to those on the PCR product.
Enzymes which lack 3´to 5´ exonuclease (proofreading) activity
are sometimes recommended to ensure accurate amplification of
the PCR product. These enzymes (such as Tli DNA polymerase)
normally generate blunt-ended PCR product.
Enzyme Concentration
It is recommended to use 1.25 units of Taq DNA polymer-
ase in a 50μl amplification reaction. For most applications, this
result in enzyme excess and the inclusion of more enzyme does
not significantly increase product yield. It should also be noted
that increased amounts of enzyme and excessively long extension
times increase the likelihood of generating artifacts associated
with the intrinsic 5´ to 3´exonuclease activity associated with Taq
DNA polymerase, resulting in smearing in agarose gels.
Pipetting errors are the most frequent cause of excessive en-
zyme levels. Accurate dispensing of sub-microliter volumes of en-
zyme solutions in 50% glycerol is nearly impossible. It is strongly
recommend to use the reaction master mix, sufficient for the
number of reactions being performed, to overcome this problem.
These master mixes will increase the initial pipetting volume of
reactants and reduce pipetting errors.
Primer Design
PCR primers generally range in length from 15–30 bases and
are designed to flank the region of interest. Primers should con-
tain 40–60% G+C and care should be taken to avoid sequences
which would produce internal secondary structure. The 3´-ends
of the primers should not be complementary to avoid the produc-
tion of primer-dimers in the PCR reaction. Avoid three G or C
nucleotides in a row near the 3´-end of the primer. Ideally, both
primers should anneal at the same temperature. The annealing
temperature is dependent upon the primer with the lowest melt-
ing temperature (Tm). The sequence of the primers can also in-
clude regions at the 5´-ends which can be useful for downstream
applications. For example, restriction enzyme sites can be placed

in the primer pair designs if the desired PCR product is to be
subsequently cloned. Regardless of primer choice, the final con-
centration of the primer in the reaction must be optimized. It is
recommend adding 50 pmol of primer (1μM final concentration
in a 50μl reaction) as a starting point for optimization.
Template Considerations
Successful amplification of the region of interest is dependent
upon the amount and quality of the template DNA. Reagents
commonly used to purify nucleic acids (salts, guanidine, proteas-
es, organic solvents and SDS) are potent inhibitors of DNA poly-
merases. Spiking a control DNA fragment and the appropriate
primer pair into the DNA preparation may be useful in verify-
ing the purity of the DNA sample. A final ethanol precipitation
of the nucleic acid sample will eliminate most of the inhibitory
agents. The amount of template required for successful amplifi-
cation is dependent upon the complexity of the DNA sample.
For example, in a 4kb plasmid containing a 1kb inserts, 25% of
the input DNA is the target of interest. Conversely, a 1kb gene
in the human genome (3.3 x 109 bp) represents approximately
0.00003% of the input DNA. Approximately 1,000,000-fold
more human genomic DNA is required to maintain the same
number of target copies per reaction. Two common mistakes are
the use of too much plasmid DNA or too little genomic DNA. If
possible, start with >104 copies of the target sequence to obtain a
signal in 25–30 cycles, but keep the final DNA concentration of
the reaction at 10ng/μl.
1μg of 1kb RNA = 1.77 x 1012 molecules
1μg of 1kb dsDNA = 9.12 x 1011 molecules
1μg of pGEM Vector DNA = 2.85 x 1011 molecules
1μg of lambda DNA = 1.9 x 1010 molecules
1μg of E. coli genomic DNA = 2 x 108 molecules
1μg of human genomic DNA = 3.04 x 105 molecules
Cycle Parameters
The sequences of the primers are a major consideration in
determining the temperature of the PCR amplification cycles.
For primers with a high Tm, it may be advantageous to increase
the annealing temperature. The higher temperature minimizes
nonspecific primer annealing, increasing the amount of specific
product produced and reducing the amount of primer-dimer
formation.Numerous formulas exist to determine the theoretical
Tm of nucleic acids, and these may serve as a starting point for an-
nealing conditions. However, it is best to optimize the annealing
conditions by performing the reaction at several temperatures,
starting approximately 5°C below any calculated Tm. The for-
mula below can be used to estimate the melting temperature for
oligonucleotides:
Tm = 81.5 + 16.6 x (log10[Na+]) + 0.41 x (%G+C) – 675/n
Where,
[Na+] is the molar salt concentration;
[K+] = [Na+] and n = number of bases in the oligonucleotides
Example:
To calculate the melting temperature of a 22mer oligonu-
cleotide with 60% G+C in 50mM KCl:
Tm = 81.5 + 16.6 x (log10 [0.05]) + 0.41 x (60) – 675/22
= 81.5 + 16.6 x (–1.30) + 24.60 – 30.68
= 53.84°C
During the extension step, allow approximately 1 minute for
every 1kb of amplicon (minimum extension time = 1 minute).
Generally, 25–40 cycles are sufficient for most reactions. Certain
unwanted side reactions can occur in PCR, and these usually be-
gin at room temperature once all reaction components are mixed.
These unwanted reactions, such as nonspecific amplification and
primer-dimer formation, can be avoided by incorporating one
of many “hot start” methods. In general, hot start techniques
limit the availability of one necessary reaction component until a
higher temperature (>60°C) is reached. This can be done manu-
ally by the addition of the critical component when the reaction

mixture reaches the higher temperature. This method, however,
is tedious and can increase the chances of contamination. Other
methods incorporate the critical substance in a wax bead, which
melts at the higher temperature, releasing the missing compo-
nent. Another technique uses an antibody to the polymerase
which, at lower temperatures, binds the polymerase, preventing
polymerization. At higher temperatures, the antibody binding is
reversed, releasing a functional polymerase.
Nucleic Acid Cross-Contamination

It is important to take great care to minimize cross-contam-
ination between samples and to prevent carryover of RNA and
DNA from one experiment to the next. Use separate work areas
and pipettors for pre- and post-amplification steps. Use positive
displacement pipets or aerosol resistant tips to reduce cross-con-
tamination during pipetting. Wear gloves and change them often.
Protocol
Materials Required
1. Template DNA
2. Downstream Oligonucleotide Primer
3. Upstream Oligonucleotide Primer
4. Taq DNA Polymerase and 10X Reaction Buffer
5. MgCl2, 25mM
6. Nuclease-Free Water
7. Nuclease-Free Light Mineral Oil
8. dNTP Mix (10mM of each dNTP)
Amplification
Note: To facilitate optimization, troubleshooting and validation
of any PCR, it is strongly recommended to include both positive
and negative control reactions.
1. Combine the first five reaction components listed in as the
table below, in a thin-walled 0.5ml reaction tube. Gently
vortex the tube for 10 seconds and briefly centrifuge in

a microcentrifuge. Initiate the reaction by adding the
template and primers.
2. Overlay the reaction with 1–2 drops (20–40μl) of
nuclease-free mineral oil to prevent condensation and
evaporation.
3. Place the tubes in a controlled temperature heat block
and proceed with the thermal cycling profile chosen for
your reactions.
Note: If working with multiple samples, master mixes consisting
of water, MgCl2, 10X Reaction Buffer, individual dNTPs, and
Taq DNA polymerase may be assembled. Combine appropriate
multiples of the listed reaction components (except template)
and add the appropriate volume such that, after template ad-
dition, the final volume is 50μl. Initiate the reaction by adding
the template. Use individual pipette tips for all additions, being
careful not to cross-contaminate the samples.
Table 1. Recommended Volumes of Components for PCR.
Components Volume Final Concentration
Nuclease-Free Water X μl
(to a final volume of 50μl)
10X Reaction Buffer 5μl 1X
dNTP mix (10mM of each dNTP) 1μl 0.2mM each
Taq DNA polymerase (5u/μl) 0.25μl 0.025u/μl
25mM MgCl2 3μl 1.5mM
Downstream Primer 50pmol1 1μM
Upstream Primer 50pmol 1
1μM
Template Yμl 2
1
A general formula for calculating the number of nanograms of primer equivalent to
50pmol is: 50pmol = 16.3ng x b; where b is the number of bases in the primer.
2
If possible, start with >104 copies of the target sequence to obtain a signal in 25–30
cycles, but keep the final DNA concentration of the reaction at ≥10ng/μl. Less than
10 copies of a target can be amplified, but more cycles may be required to detect a
signal by gel electrophoresis. Additional cycles may increase nonspecific amplification,
evidenced by smeared bands upon gel electrophoresis.
Analysis
1. Analyze the PCR reaction products by agarose gel elec-
trophoresis of a 5μl aliquot from the total reaction. The
products should be readily visible by UV transillumina-
tion of the ethidium bromide-stained gel.
2. Store reaction products at -20°C until needed. The reac-
tion products can be further purified using a DNA Puri-
fication System.
Troubleshooting PCR
Symptoms Possible Causes Comments
Comments
Low yield Insufficient Return reactions to thermal cycler for 5 or
or no am- number of cycles more cycles.
plification Template de- Verify the integrity of the DNA by elec-
Product graded trophoresis after incubation in the pres-
ence of Mg2+.
Thermal cycler Verify that time and temperatures are cor-
programmed rect. Use step cycles, not hold segments.
incorrectly
Temperature Perform a set of control reactions to de-
too low in some termine if certain positions in the thermal
positions of cycler give low yields.
thermal cycler
Top of thermal The top must be closed for correct heating
cycler open and cooling.
Inhibitor present Reduce the volume of sample in the re-
action. Ethanol precipitate to remove in-
hibitors.
Improper reac- Reduce the annealing temperature or al-
tion conditions low longer extension times for longer am-
plicons.
Mineral oil The reaction must be overlaid with high
problem quality, nuclease-free light mineral oil. Do
not use autoclaved mineral oil.
Poor primer Make sure primers are not self-comple-
design mentary or complementary to each other.
Try a longer primer.
Incorrect primer Verify that the primers are complemen-

specificity tary to the appropriate strands.
Primer concen- Verify primer concentration in the reac-
tration too low tion. Increase primer concentration in the
reaction
Suboptimal reac- Optimize Mg2+ concentration, annealing
tion conditions temperature and extension time. Always
vortex the Mg2+. Store solution prior to
use. Verify that primers are present in
equal concentration.
Nucleotides Keep nucleotides frozen in aliquots, thaw
degraded quickly and keep on ice once thawed.
Avoid multiple freeze/thaw cycles.
Target sequence Redesign experiment or try other sources
genuinely not of target DNA.
present in target
DNA
Primer annealing If oligo (dT) was used as a downstream
primer, verify that the annealing incuba-
tion was carried out at an appropriate
temperature.
Primer specific- Verify that the downstream primer se-
ity quence is complementary to the down-
stream sequence of the RNA.
References
1. Erlich, H.A. 1989. PCR technology: principles and applications
for DNA amplifications. Stockton Press, NY.
2. Newton, C.R. and A. Graham. 1994. PCR, part 1: Basic
principles and methods. EngBios Scientific Publishers, Oxford.
3. Saiki, R.K., D.H. Gelfand, S. Stoffel, S.J. Scharf, R. Higuchi,
G.T. Horn, K.B. Mullis and H.A. Erlich. 1988. Primer-
directed enzymatic amplification of DNA with a thermostable
DNA polymerase. Science 239:487-491.
4. Higuchi R (1989) Simple and rapid preparation of samples
for PCR. In: HA Erlich (ed): PCR technology, principles and
applications for DNA amplification,Stockton Press, New York,
31-38.
Isolation of Total RNA from Bacterial Cells 61
10.
Isolation of Total RNA from Bacterial
Cells
Principle
The endogenous RNase level varies with cell type, thus necessary
precautions will vary. These may include the use of guanidinium
thiocyanate (GuSCN), phenol, a thiol reagent (ß-mercaptoeth-
anol, dithiothreitol), proteinase K, a detergent (sodium dode-
cyl [lauryl] sulphate, N-dodecyl sarkosine [sarkosyl]), placental
RNase inhibitor, and vanadyl ribonucleoside complexes. Some of
the reagents (phenol, detergent, proteinase K, GuSCN) will also
simultaneously deproteinise RNA.
Following the addition of GuSCN, RNA may be separated
from protein and DNA using phenol prior to precipitation with
ethanol or isopropanol, although in extracting rRNA phenol
treatment is not necessary. The pH must not be alkaline in view
of the ability of RNA, and is normally 7.0± 0.2. Phenol treat-
ment may be with buffer-saturated phenol alone, or with phe-
nol-chloroform-isoamyl alcohol (25:24:1) in which subsequent
phase separation is easier but both yield RNA in the upper, aque-
ous layer. Chloroform alone, without phenol, has been recom-
mended for bacterial RNA extraction.
Polyethylene glycol is used to precipitate intact virus particles
and plasmid DNA during plasmid or viral DNA preparation. It has
been found that, following cell disruption, the cell debris and DNA
can be removed by the addition of polyethylene glycol and salts,
leaving the RNA in solution. Subsequently, an aqueous biphasic
system may be produced by the addition of more salt, in which

RNA selectively partitions into the lower, salt phase. Whether this
is done or not, the RNA may then be precipitated with or without
prior phenol treatment.
Materials
All solutions are prepared in water that has been previously
autoclaved with 0.1% (v/v) DEPC. The latter is suspected of be-
ing a carcinogen and should be handled with care. Note: Care
should be exercised when handling GuSCN and SDS, particu-
larly in the solid state.
1. SDS 5% (w/v).
2. Guanidinium thiocyanate (GuSCN) 6M. Filter and store
at room temperature.
3. Guanidinium thiocyanate 4M.
4. Sodium citrate 25 mM, pH 7.0.
5. ß-mercaptoethanol.
6. Proteinase K: 0.5 mg/mL. Aliquot and store at -20°C.
7. Physiological saline, 0.85% (w/v) NaCl. Autoclave and
8. Polyethylene glycol 6000 (PEG) 20% (w/v), 0.75% SDS
(w/v) in 7.5% (w/v) potassium phosphate, pH 7.2. Auto-
clave and store at room temperature.
9. Polyethylene glycol 6000 (PEG): 12.5% (w/v) in 1%
(w/v) potassium phosphate, pH 7.2
10. 0.12 M Sodium phosphate buffer, pH 7.2
11. Phenol, saturated with 0.12 M phosphate buffer, pH
7.2. To phenol (either freshly redistilled at 165-180°C,
or provided for use in molecular biology) in a previously
unopened bottle, add phosphate buffer until the bottle is
full. Mix gently. Add 0.05% (w/w of phenol) 8-hydrox-
yquinoline as an antioxidant and allow phase separation
at 4°C.Remove and discard the upper, aqueous layer and
repeat the buffer addition, mixing and decanting. Store
the phenol at -20°C.

Note: Phenol burns the skin. Should this occur, wash
with 20% (w/v) PEG in 50% (v/v) industrial methylated
spirits.
12. Chloroform.
13. Sodium acetate, 3M, pH 5.2.
14. TBE buffer: Tris borate EDTA buffer: 90 mM Tris (hy-
droxymethyl) aminomethane, 90mM boric acid, 2.5mM
EDTA, pH 8.3. Prepare a stock 1OX solution by dissolv-
ing 108 gm Tris base, 55gm boric acid, and 9.5gm diso-
dium EDTA in water, to 1L.
15. Agarose, 1.1% (w/v) in TBE. Agarose should be low elec-
tro-endosmosis (EEO).
16. TE buffer: 10mM Tris-HCI, pH 8.0, 1mM EDTA. Au-
toclave.
17. 10% (w/v) Sodium dodecyl sulphate. Autoclave.
18. L-broth: 10 gm/L Tryptone, 5 gm/L yeast extract, 5 gm/L
NaCl, 1gm/L glucose.
Procedure
A. Bacterial Culture
1. Inoculate 50 mL of LB-broth and incubate overnight at
37°C having an absorbance of 0.45-0.60 at 600 nm.
2. Centrifuge a 1.5 mL sample in an eppendorf tube at
12,000 rpm for 10 min. Discard the supernatant.
3. Wash in saline by resuspending the pellet in 300μL of
saline, centrifuge at 12,000 rpm for10 min and discard
the supernatant.
4. Resuspend the pellet in 400 μL of 0.12 M sodium phos-
phate buffer, pH 7.2
B. Lysis and Extraction with PEG 6000

1. To the resuspended pellet add 50 μL of 5% SDS and 50
μL of proteinase K (0.5 mg/mL).Vortex and incubate at
37°C for 20 min.

2. Add 500 μL of PEG 6000 (12.5% w/v) in 1% potassium
phosphate, pH 7.2. Vortex and centrifuge at 12,000 rpm
for 10 min.
3. Carefully remove the supernatant, measuring the volume,
into a sterile tube for precipitation of the RNA. If neces-
sary, phenol extraction may be carried out at this stage.
4. Add 0.1 vol of 3M sodium acetate, pH 5.2, followed by
2.5 volume of ethanol (or 1 volume of isopropanol) at-
20°C to precipitate the RNA. Leave for at least 1hr.
5. Centrifuge at 12,000
rs TMum
rpm for 15 min, discard ke i
ar nn
d
the supernatant and
M ille
de
ra
ct
M
eg
ta
drain the tube by care-
In
D
fully inverting onto tis- 9
6
sue paper. Wash the pel- 54 23S
let (which may not be 32.5
visible) with 70% etha- 21.5 16S
nol by resuspending and 1
centrifuging at 12,000 0.5
rpm for 10 min. Decant
and drain as before.
6. Dissolve the pellet in Fig: Agarose gel electrophoresis of
TE at room tempera- intact 23S and 16S rRNA.
ture for at least 30min.
The yield is about 15 μg total RNA/mL of culture. The
ratio A260/A280 is in the range 1.90-2.05.
7. Should protein removal by phenol be required, add, se-
quentially, 0.1 volume of 3M sodium acetate, pH 5.2 and
1 volume buffer-saturated phenol. Vortex and centrifuge
at 12,000 rpm for 10 min. Remove the upper, aqueous
layer, repeat the extraction of this layer with 1 volume of
phenol, vortex and centrifuge and retain the upper layer.
The RNA may be precipitated without adding more so-
dium acetate.
C. Lysis and Extraction with GuSCN

I. Addition after Cell Lysis
1. Cells are prepared and lysed.
2. Add 75 μL of 6 M GuSCN, vortex and centrifuge at
12,000 rpm for 10 min. Transfer the supernatant to a
fresh tube.
3. RNA can be precipitated directly at this stage, without
significant protein contamination, by the addition of 0.1
volume of sodium acetate and 2.5 volumes of cold etha-
nol.
4. Alternatively, phenol extraction may be carried out prior
to precipitation.
II. Addition before Cell Lysis

Bacteria can be lysed by brief sonication in the presence of
GuSCN. The procedure subsequently is then the one-step acid
GuSCN-phenol method, except that the detergent is added after
sonication to avoid frothing.
1. Resuspend the bacterial pellet in I ml of 4M GuSCN +
sodium citrate + ß-mercaptoethanol and sonicate for 20
sec.
2. Add 50 μL of 10% sarkosyl, vortex and centrifuge at
12,000 rpm for 10 min.
3. The RNA may be precipitated at this stage by the addi-
tion of 0.1 volume of 2 M sodium acetate, pH 4.0 and
either 2.5 volume of ethanol or 1 volume of isopropanol
at -20°C followed by centrifugation at 12,000 rpm for
10min, or phenol-extracted in the presence of acidic so-
dium acetate and acidic phenol.
4. Add, sequentially, 0.1 volume of 2M sodium acetate, pH
4.0, 1 volume of water –saturated phenol and 0.2 volume
of chloroform isoamyl alcohol (49: 1, v/v), vortex and
centrifuge at 12,000 rpm for 10min.
5. The RNA in the upper, aqueous phase is precipitated by
the addition of 2.5 volumes of ethanol or 1 volume of

isopropanol at -200C.
Precautions
1. Some organisms (Pseudomonas aeruginosa, K. aerogenes)
may be extracted under the conditions given, whereas
others that have been tried (Salmonella typyhimurium,
Proteus mirabilis, Serratia marcescens) will require changes
in concentrations of some reagents, in particular SDS
and/or PEG may be increased.
2. If the RNA is not to be precipitated and washed, and if
small amounts of phenol interfere with subsequent treat-
ment of the RNA, then phenol can be removed by wash-
ing with chloroform. Add about an equal volume of chlo-
roform, mix and centrifuge very briefly, discard the lower,
chloroform layer. Repeat at least four times. Phenol may
affect enzyme activity and certainly will give spuriously
high A260/A280 ratio since it gives a peak at 270 nm.
3. The efficacy of GuSCN as an inhibitor of RNase is de-
pendent on the concentrations of both inhibitor and
enzyme. Chaotropic effects are not apparent below 3M
GuSCN; in some cases 5M may be required for sufficient
inhibition.
References
1. Chomczynski P, Sacchi N. 1987. Single step method of RNA
isolation by acid guanidinium iso thiocyanate-phenolchloro-
form extraction. Analytical Biochemistry, 162, 156-159.
2. Puissant, C. and L.M. Houdebine. 1990. An Improvement
of the Single-Step Method of RNA Isolation by Acid Guani-
dinium Thiocyanate-Phenol-Chloroform Extraction. Bio-
Techniques 8: 148 – 149.
3. Sambrook J, Fritsch EF, and Maniatis T. 1989. Molecular
Cloning: A Laboratory Manual. 2nd edition. New York:
Cold Spring Harbor Laboratory Press.
Total RNA Isolation with TRIZOL Reagent 67
11.
Total RNA Isolation with TRIZOL
Reagent
Principle
TRIZOL Reagent is a ready-to-use reagent for the isolation of
total RNA from cells and tissues. The reagent, a mono-phasic
solution of phenol and guanidine isothiocyanate, is an improve-
ment to the single-step RNA isolation method. During sample
homogenization or lysis, TRIZOL Reagent maintains the integ-
rity of the RNA, while disrupting cells and dissolving cell com-
ponents. Addition of chloroform followed by centrifugation sep-
arates the solution into an aqueous phase and an organic phase.
RNA remains exclusively in the aqueous phase. After transfer of
the aqueous phase, the RNA is recovered by precipitation with
isopropyl alcohol.
Reagents
1. DEPC-treated water
2. TRIZOL Reagent
3. Ice cold PBS
4. Cell scraper
5. 70% ethanol
6. Isopropyl alcohol
Homogenization
1. Tissues:
Homogenize tissue samples in 1 ml of TRIZOL reagent per
50 to 100 mg of tissue using a glass-teflon or power homogenizer
(tissue lyser). The sample volume should not exceed 10% of the
volume of TRIZOL Reagent used for the homogenization.
2. Cells grown in Monolayer
Rinse the cell monolayer with ice cold PBS once. Lyse cells
directly in a culture dish by adding 1 ml of TRIZOL Reagent
per 3.5 cm diameter dish and scraping with cell scraper. Pass the
cell lysate several times through a pipette; vortex thoroughly. The
amount of TRIZOL reagent added is based on the area of the
culture dish (1 ml/10cm2) and not on the number of cells pre-
sent. An insufficient amount of TRIZOL Reagent may result in
DNA contamination of the isolated RNA.
3. Cells Grown in suspension

Spin cells for 5 min at 500 rpm. Remove media and resus-
pend cells in ice cold PBS. Pellet cells by spinning at 500 rpm for
5 min. Lyse cells with TRIZOL reagent by repetitive pipetting or
by passing through syringe and needle. Use 1 ml of the reagent
per 5-10 x 106 of animal cells. Incubate the homogenized sample
for 5 minutes at room temperature to permit the complete dis-
sociation of nucleoprotein complexes. Centrifuge to remove the
cell debris. Transfer the supernatant to new tube.
4. Phase Seperation
Add 0.2 ml of chloroform per 1 ml of TRIZOL Reagent.
Cap sample tubes securely. Vortex samples vigorously for 15
seconds and incubate them at room temperature for 3 minutes.
Centrifuge the samples at 12,000 rpm for 15 minutes at 40C.
Following centrifugation, the mixture separates into lower red,
phenol chloroform phase, an interphase, and a colourless upper
aqueous phase. RNA remains exclusively in the aqueous phase.
Transfer upper aqueous phase carefully without disturbing the
Total RNA Isolation with TRIZOL Reagent 69
interphase into fresh tube. Measure the volume of the aqueous

phase (the volume of the aqueous phase is about 60% of the vol-
ume of TRIZOL Reagent used for homogenization).
5. RNA Precipitation
Precipitate the RNA from the aqueous phase by mixing with
isopropyl alcohol. Use 0.5ml of isopropyl alcohol per 1 ml of
TRIZOL reagent used for the initial homogenization. Incubate
samples at 25oC for 10 minutes and centrifuge at 12,000 rpm for
10 minutes at 4oC. The RNA precipitate, often invisible before
centrifugation, forms a gel-like pellet on the side and bottom of
the tube.
6. RNA Wash
Remove the supernatant completely. Wash the RNA pellet
once with 75% ethanol, adding at least 1 ml of 75% ethanol
per 1 ml of Trizol reagent used for the initial homogenization.
Mix the samples by vortexing and centrifuge at 7,500 rpm for 5
minutes at 80C. Repeat above washing procedure once. Remove
all left over ethanol.
7. Redissolving RNA
Air-dry the RNA pellet for 5-10 minutes. Do not dry the
RNA pellet by centrifuge under vacuum. It is important not to
let the RNA pellet dry completely as this will greatly decrease its
solubility. Partially dissolved RNA samples have an A260/A280ratio
< 1.6. Dissolve RNA in DEPC-treated water by passing solution
a few times through a pipette tip.
8. Spectrophotometric Analysis
Dilute 1 μl of RNA with 39 μl of DEPC-treated water (1:40
dilution). Using the 10 μl microcuvette, take OD at 260 nm and
280 nm to determine sample concentration and purity. The A260/
A280 ratio should be above 1.6. Apply the convention that 1 OD
at 260 equals 40 µg/ml RNA.
Precautions
1. Always wear gloves and lab coat while performing the ex-
periment.
2. Avoid contact with skin or clothing.
3. Use in a chemical hood. Avoid breathing vapours.
References
1. Puissant, C. and L.M. Houdebine. 1990. An improvement
of the single-step method of RNA isolation by acid guanidin-
ium thiocyanate-phenol-chloroform extraction”. BioTech-
niques 8: 148– 149.
2. General remarks on handling RNA. The Bench Guide. Qia-
gen.
3. Chomczynski P. 1993. A reagent for the single-step simul-
taneous isolation of RNA, DNA and proteins from cell and
tissue. Biotechniques 15, 532-537.
4. RNA isolation Protocols Website
http://www.nwfsc.noaa.gov/protocols/methodslR-
NAMethodsMenu.html
Extraction of Total Plant RNA 71
12.
Extraction of Total Plant RNA
Principle
Plants are diverse, and individual species and organs or tissues of
plants can behave differently during extraction of RNA for use in
molecular studies. Hence, a range of extraction methods has also
been devised, depending on the tissue or genotype being extracted.
Problems encountered include the presence of large quantities
of polysaccharides; high levels of RNases; various different kinds
of phenolics, including tannins; low concentrations of nucleic
acids (high water content); tissue, such as lignin (wood), that is
difficult to break up; and so on. In addition, sampling techniques
can have an effect on yield and lack of degradation, recognizing
also that most tissues extracted are generally composed of a
range of cell types and, hence, functions. In some instances,
commercial kits are sufficient to perform the task, but, in other
instances, especially with a new plant or tissue that has not had
RNA extracted from it before, methods may need to be modified
to suit the particular characteristics of that material. There is no
simple indication that a tissue will be difficult. Depending on the
use of the RNA extracted, further purification of messenger RNA
(mRNA) may be required. The biggest problem encountered in
RNA extraction usually originates from the initial sampling and
extraction protocols. Three extraction protocols will, therefore,
be outlined that have had wide spread success in plant RNA
extraction.
Materials And Method

1. Sampling
1. Liquid nitrogen
2. Polystyrene box
3. Sharp knife, scalpel, razor blade, tweezers, cork borer,
metal needle/probe, and flame source
4. Eppendorf tubes, tinfoil, and plastic bottles of various
sizes
5. Analytical balance
6. Plant material
7. –80°C freezer or liquid nitrogen storage container
Sampling Method
In general, tissue from a plant is sampled by plucking and
covering immediately with liquid nitrogen in a polystyrene con-
tainer. It is essential to have a minimal time between removal
from the plant and immersion in liquid nitrogen, to minimize
the expression of new mRNAs because of tissue wounding or
detachment from the plant. Some mRNA has been shown to be
upregulated within 5 min. of tissue detachment from a plant.
Other tissues are more bulky, e.g., fruit or tubers; these tissues
take longer to equilibrate to liquid nitrogen temperatures if im-
mersed whole. Bulky tissues hold more heat, and exchange is
slower with liquid nitrogen. This leads to tissue damage and
degradation of the mRNA present, because RNases gain ac-
cess to the mRNA. Hence, for bulky tissues, it is better to rap-
idly remove the tissue from the plant and to subsample quickly
(preferably a minute between detachment and immersion of
subsamples in liquid nitrogen). Tissue samples can be added
directly to preweighed eppendorf tubes or storage containers.
If using eppendorf tubes, prepare the tubes with a small hole
(heat a needle over a flame and pierce the lid) to prevent ex-
plosions because of the remnants of liquid nitrogen inside the
sealed tube when the lids are closed. This can also be done with
other containers, or else the container can be drained before
placing the lid on the container. After the tissue has been sam-
pled, store the sample in a liquid nitrogen storage container or
a –80°C freezer. To extract RNA, grind the weighed material in
a mortar and pestle, under liquid nitrogen, to a fine powder and
transfer the powder to the extraction buffer.
Note: do not ever let the plant tissue thaw after inserting the tis-
sue in liquid nitrogen and before complete mixing in extraction
buffers after grinding the tissue to a powder. The amount of tis-
sue required to achieve acceptable yields of RNA varies according
to the material. Tissues with high water content require higher
amounts of tissue to be extracted. For example, 2 gm Arabidopsis
leaf tissue yields approximately 60 to 200 μg RNA (Trizol meth-
od); and 5 to 8 gm fruit tissue (high water) yields approx 400 μg
RNA (non-CTAB/non-guanidine-based method).
Plant RNA Extraction

A.Trizol or Guanidine Isothiocyanate-Based Method
Reagents
1. Trizol reagent
2. Chloroform
3. Isopropyl alcohol
4. 75% ethanol in RNase-free water
5. RNase-free water (made by adding 0.01% DEPC [v/v],
standing or stirring overnight, then autoclaving
6. 0.1M NaOH-washed and UV-treated plastic ware, oven-
baked sterile glassware, sterilized eppendorf tubes
7. Liquid nitrogen and mortar and pestle
8. Benchtop centrifuges
9. Commercial mRNA purification kit
Method
1. Grind 1gm plant tissue in liquid nitrogen.
2. Add 1 mL of Trizol reagent to the ground powder.
3. Transfer into 2 ml eppendorf tubes.
4. Centrifuge at 12,000 rpm for 5 min at 4°C.

5. Transfer the supernatant to new eppendorf tube.
6. Add 200 μL of chloroform and shake vigorously for 15
sec.
7. Incubate at room temperature for 5 min.
9. Carefully transfer the upper aqueous phase to a new ep-
pendorf tube (ensure no interface debris is transferred).
10. Add 0.5 mL of isopropyl alcohol. Mix well.
11. Incubate at room temperature for 10 min.
12. Centrifuge at 12,000 rpm for 10 min, at 4°C.
13. Carefully discard supernatant (tip eppendorf tube with
the pellet position angled up and away from you and pi-
pette out the supernatant).
14. Vortex briefly and centrifuge at 12,000 rpm for 5 min at
4°C.
15. Discard the supernatant as in step 13 and allow pellet to
air-dry for 10 min.
16. Dissolve the pellet in 20 μL of RNase-free water by very
gently sucking the liquid up and down with a pipette.
17. Quantify the RNA, check the purity and degradation,
and either store at –20 or –80°C until used, or extract the
mRNA using commercial kits.
B. Extractions from Problem Tissues: CTAB Method

Materials
1. Sterile falcon tubes (conical bottom, 25 or 50 mL).
2. Oakridge tubes (round bottom, sterile, and RNase-free).
3. Mira-Cloth.
4. Liquid nitrogen and mortar and pestle.
5. Benchtop centrifuge, vortex machine, refrigerator, and
freezer.
6. RNase-free water in a baked storage bottle (in an oven at
>150°C, for >4 hr).
7. Chloroform: isoamyl alcohol (24:1).

8. 12M LiCl.
9. Extraction buffer: 2% CTAB; 2% PVPP; 100mM Tris-
HCl, pH 8.0; 25mM EDTA, pH 8.0; 2M NaCl; 0.5 gm/L
spermidine; and 2% β-mercaptoethanol. Use RNase-free
water for dissolving, and autoclave before using.
10. SDS–Tris-HCl–EDTA (SSTE) buffer: 1M NaCl; 0.5%
SDS; 10mM Tris-HCl, pH 8.0; 1mM EDTA, pH 8.0.
Use RNase-free water and autoclave before using.
Method
1. Pipette 15 mL of extraction buffer (without
β-mercaptoethanol) into an RNase-free falcon tube and
add 300 μL of β-mercaptoethanol. Incubate in a water
bath at 65°C for 10 min.
2. Grind the tissue in liquid nitrogen and add the tissue
gradually to the heated buffer so that no powder coagu-
lates; vortex to ensure that the powder is fully dispersed.
3. Leave the sample at room temperature while processing
the next samples.
4. Homogenize the samples for 1 min at full speed until the
sample foams close to the top of the tube. Wash the ho-
mogenizer with distilled water after each sample.
5. Add an equal volume of chloroform: isoamyl alcohol mix
(vortex), transfer to an RNase-free oakridge tube, balance
the samples with buffer, and centrifuge at 12000 rpm for
10 min at room temperature to separate the phases.
6. Filter the upper aqueous phase through an autoclaved
Mira-cloth into a new RNase-free oakridge tube (or care-
fully pipet off the top aqueous phase, ensuring no transfer
of any interface material to a new tube).
Note: It is better to leave some aqueous phase behind
than to transfer contaminants.
7. Add an equal volume of chloroform: isoamyl alcohol,
mix, and centrifuge as in step 5 to separate the phases.
8. Transfer the top aqueous phase to an RNase-free falcon

tube and estimate the volume. Add an appropriate vol-
ume of LiCl solution to give a final concentration of 2M
LiCl (1 volume of 4 M, 0.5 volumes of 6 M, 0.33 vol-
umes of 8 M, 0.25 volumes of 10 M, or 0.2 volumes of
12 M).
9. Leave at 5°C (refrigerator) overnight.
10. Centrifuge at 12000 rpm for 20 min at 4°C.
11. Pour off the supernatant.
12. Preheat SSTE buffer to 65°C. Dissolve the pellet in 200
μL of heated SSTE, and transfer to a 1.5 mL, RNase-free
eppendorf tube.
13. If the SDS in the SSTE buffer precipitates, place the ep-
pendorf with the sample in a heating block (37°C) until
it has dissolved again.
14. Add an equal volume of chloroform: isoamyl alcohol;
vortex immediately.
15. Add 2 volumes of absolute ethanol to precipitate RNA
(>30 min at –70°C, or >2 hr at –20°C).
16. Centrifuge the tube in a microcentrifuge at 4°C for 20
min at maximum speed.
17. Discard the supernatant, allow the pellet to air-dry, and
resuspend in 20 μL RNase free water.
C. E
xtractions from Problem Tissues: Non-CTAB-Based
or Non-Guanidine-Based Method
Materials
1. Eppendorf/Falcon tubes (sterile and RNase-free).
2. Liquid nitrogen.
3. Mortar and pestle.
4. Homogenizer.
5. Oakridge tubes (sterile and RNase-free) and Corex tubes
(sterile and RNase-free).
6. Benchtop centrifuge, vortex machine, refrigerator, and

freezer.
7. RNase-free water in a baked storage bottle (in an oven at
>150°C, for >4 hr).
8. Preheated (65°C) lysis buffer: 150mM Tris-HCl, 50mM
EDTA, 4% SDS, pH 7.5 titrated with boric acid, 1%
β-mercaptoethanol, and 1% w/w PVPP. Use RNase-free
water and autoclave before use.
9. 5M potassium acetate; use RNase-free water and auto-
clave.
10. Absolute ethanol chilled.
11. Chloroform: isoamyl alcohol (24:1).
12. Tris-HCl-equilibrated phenol, pH 8.0. Keep phenol in
dark bottles in –20°C. Make the Tris-HCl buffer RNase-
free by adding DEPC to make buffer in a sterile bottle, do
not autoclave buffer.
13. 12M LiCl (use RNase-free water and autoclave).
Method
1. Very slowly add 5 gm of ground powder to 15 mL of
preheated lysis buffer containing PVPP and freshly added
β-mercaptoethanol, and vortex simultaneously (do not
allow powder to thaw or form lumps).
2. Homogenize the suspension at maximum speed for 20
sec, or until the froth reaches the top of the tube.
3. Add 0.1 volumes (1.5 mL) of 5M potassium acetate and
0.25 volumes (4 mL) of chilled absolute ethanol to the
tube and vortex for 30 sec.
4. Add 1 volume of chloroform: isoamyl alcohol to each of
two oakridge tubes and put half of the homogenate in
each tube, vortex, and centrifuge at 2000 rpm for 10 min
at room temperature.
5. Transfer the top aqueous phase to an RNase-free falcon
tube and add 10 mL of buffered phenol and 10 mL of
chloroform: isoamyl alcohol.
6. Vortex to mix, and centrifuge at 2000 rpm for 10 min to

separate the phases.
7. Repeat steps 5 and 6.
8. Transfer the top aqueous phase to an oakridge tube and
add one-third volume of 12M LiCl. Incubate overnight
at –20°C.
9. Centrifuge at 20,000 rpm for 20 min to precipitate the
pellet.
10. Pour off the supernatant and resuspend the pellet (by vor-
texing) in 10 mL of 3M LiCl (12M LiCl diluted with
RNase-free water). Centrifuge at 20,000 rpm for 20 min.
11. Pour off the supernatant, and resuspend the pellet (by
vortexing) in 2 mL of RNase-free water, transfer to a 30-
mL corex tube.
12. Add 180 μL of 5M potassium acetate and 6 mL of chilled
absolute ethanol. Cover with parafilm and leave at –20°C
for 1 h.
14. Pour off the supernatant and dry pellet in air for 10 min.
15. Dissolve the pellet in 200 μL of sterile RNase-free water;
store at –20°C.
D. Quantification, Degradation and Storage

Materials
1. RNase-free water.
2. 10X stock Tris-base–boric acid–EDTA buffer: 108gm
Tris-base, 55 gm boric acid, and 40 mL of 0.5M EDTA,
pH 8.0, in 1 L deionised water.
3. 37% formaldehyde.
4. Agarose (Low EEO).
5. 10X MOPS buffer: 0.2M MOPS (3-[N-morpholino]
propane sulfonic acid, 50mM sodium acetate, and 10mM
EDTA).
6. Loading buffer (store in aliquots at –20°C): 0.75 mL
deionised formamide, 0.15mL of 10X MOPS buffer

(autoclaved), 0.24 mL formaldehyde, 0.1 ml RNase-free
water, 0.1 ml glycerol (autoclaved), and 10% w/v bromo-
phenol blue. Add 3 μL ethidium bromide to 300 μL load-
ing buffer before using.
7. Ethidium bromide 10% solution.
8. RNase-free electrophoresis gel boxes, beds, and combs.
Method
1. To quantify and assess the degree of purity, take a 2.5 μL
aliquot and dilute with 1 mL of water. Scan in a UV-spec-
trophotometer from 190 nm to 320 nm. Alternatively,
read in a spectrophotometer at 260 and 280 nm.
Calculate the concentration of RNA by the formula:
OD260 × dilution factor/25; 1 ×OD260 =40 μg/mL RNA.
2. To assess whether extracted RNA is degraded and to con-
firm the quantification, run an aliquot on an agarose gel.
Place 2.5 μL of RNA in an eppendorf tube and add 10 μL
loading buffer. Prepare the gel apparatus; soak the appa-
ratus for at least 1 h in water plus SDS (10%) to denature
any RNases. Rinse in RNase-free water. Prepare a 1% for-
mamide agarose gel. For a 30-mL gel, take 3 mL of 10X
MOPS buffer, add 25.3 mL RNase-free water and 0.3 gm
agarose; heat in a microwave oven for 35 sec and add 1.7
mL of 37% formaldehyde. Pour the solution from step 5
into the gel apparatus and wait until it solidifies. Remove
the combs. Add 200 mL of 1X MOPS running buffer to
cover the gel and wells. Pre-equilibrate gel by running at
80 V for 10 min. Add 2 volumes of RNA loading buffer
to 1 volume of sample. Heat at 65°C for 10 min. Rinse
wells with buffer, and load the RNA samples into lanes.
Load one lane with 5 μL or the recommended quantity of
an RNA standard. Run the gel for 1.5 h at 80 V. Visualize
under UV light.
3. RNA degradation (or contamination) can be detected by:
(a) A blob at the running edge end of the gel—the RNA

is totally degraded.
(b) A smear with indistinct bands present (this can also
mean there is a lot of polysaccharide in the sample).
(c) The two main ribosomal bands are equal in intensity,
or the lower band is higher than the upper band.
(d) There is a bright band at the top of the gel near the
loading wells indicating the presence of DNA in the
sample. Depending on the use of the RNA, this may
not be a problem. It can be removed by digesting
with an RNase-free DNase.
References
1. Salzman, R.A. et al. 1999. An improved RNA isolation
method for plant tissues containing high levels of phenolic
compounds or carbohydrates. Plant Molecular Biology Re-
ports 17, 11-17.
2. Sambrook J and Russell DW. 2001. Molecular cloning. A
laboratory manual. 3rd edition, Cold Spring Harbor Labo-
ratory Press, Cold Spring Harbor, New York.
3. Chang, S., Puryear, J., and Cairney, J. 1993. A simple and
efficient method for isolating RNA from pine trees. Plant
Molecular Biology Reports 11, 113–116.
4. Manning, K. 2000. Isolation of nucleic acid from plants by
differential solvent precipitation. Analytical Biochemistry,
195, 45-50.
5. Meisel, L. et al. 2005. A rapid and efficient method for puri-
fying high quality total RNA from peaches (Prunus persica)
for functional genomics analyses. Biological Research, 38,
83-88.
6. Sharma, AD. et al. 2003. RNA isolation from plant tissue
rich in polysaccharides. Analytical Biochemistry, 314, 319-
321.
Total Protein Extraction with TCA-Acetone and 2D-Gel Electrophoresis 81
13.
Total Protein Extraction with TCA-
Acetone and 2D-Gel Electrophoresis
Introduction
In the context of proteomic studies, comparison of 2D gels re-
quires well resolved proteins; streaking and smearing must be
avoided, as well as artifacts caused by proteolysis. Protein pat-
terns must be reproducible from gel to gel. Sample preparation is
thus a crucial step prior to electrophoresis. The main difficulties
with plant tissues are low cellular protein content, the presence
of proteases and interfering compounds such as phenolics, pig-
ments, lipids and nucleic acids. After extracting as many differ-
ent proteins as possible, one has to solubilize them in a solution
compatible with isoelectric focusing (IEF).
After working with a large variety of plant tissues, a method
in which proteins are denatured and precipitated in a mixture of
ß-mercaptoethanol and trichloracetic acid (TCA) in cold acetone.
TCA precipitation efficiently inhibits protease activity in plant tis-
sues. Proteins were then solubilized in the urea-K2CO3 solution.
This procedure gives highly reproducible gels with a good spot res-
olution in large pH and Mr ranges, and it has become very popular
under the name of the TCA/acetone method. The solubilization
solution was developed for the first dimension in IPG strips. How-
ever, running protein samples on IPGs prompted modifications
in solubilization procedures, because of high salt content and the
presence of ionic detergent (SDS) that are not compatible with the
high voltages required to perform separation in such gels.
Materials
1. Precipitation solution: 10% TCA (w/v), 0.07% ß-ME
(v/v) in cold acetone. This solution must be freshly pre-
pared and stored at –20°C until use.
2. Rinsing solution: 0.07% ß-ME (v/v) in cold acetone.
This solution can be stored at –20°C for about 1 month.
3. Solubilization solution: 9.5M urea, 5mM K2CO3, 1.25%
SDS, 5% DTT, 6% Triton X-100, 2% ampholines 3.5 to
9.5 in DDW. K2CO3is prepared as a 2.8 % (w/v) stock
solution and SDS as a 10% (w/v) filtered stock solution,
Triton X-100 is provided as a 20% solution. Then 3 mL
DDW are added to the other components until the urea
is solubilized. Solutions of 40 mL are usually prepared
and then aliquoted and stored at –80°C.
4. Determination of protein concentration: The protein
content of the samples is evaluated by Bradford’s assay
(discussed in next chapter). The procedure is compatible
with common sample preparation reagents.
5. IPG strip rehydration solution: 7M urea, 2M thiourea,
1.4% CHAPS (w/v), 16mM DTT, 5mM phosphine,
0.3% ampholyte 3-10 (v/v) in DDW.
Method
A. Protein Precipitation and Denaturation
1. Grind the plant tissues in a mortar and pestle in liquid
nitrogen to obtain a fine powder.
2. Transfer about 200 μL of powder to a weighed 2-mL
eppendorf tube. Cover with 1.8 mL of the cold TCA-
ßME-acetone solution, mix and store at –20°C for 1 h.
The solution inactivates the phenoloxidases and oxidases,
preventing phenol oxidation into quinones, which would
result in protein binding into insoluble complexes. It has
also been shown to inactivate proteases. Acetone alone al-
lows solubilization of the pigments, lipids, and terpenoids

possibly present in the tissue. ß-ME prevents the forma-
tion of disulfide bonds during precipitation.
3. Centrifuge for 10 min at 10,000 rpm (in a refrigerated
centrifuge, below 4°C).
B. Rinsing with ß-ME-Acetone Solution

1. Discard the supernatant and resuspend the pellet in 1.8
mL of cold rinsing solution. Store at –20°C for 1 h. This
step eliminates the acidity caused by TCA that would im-
pede protein recovery.
2. Centrifuge for 15 min at 10,000 rpm (in a refrigerated
centrifuge, below 4°C).
3. Discard the supernatant. This step is repeated twice (see
Note 6).
4. Dry the pellet under vacuum for about 1 h to eliminate
the acetone fully (alternatively, dry for 20–30 min in a
SpeedVac without heating).
5. Weigh the pellet (see Note 7).
C. Protein Solubilization
1. The amount of buffers for protein solubilization depends
on the plant tissue; for example, use 60 μL/mg dry pow-
der for leaf tissue (maize, rape) and 50 μL/mg dry powder
for maize kernels.
2. Resolubilization is achieved by vortexing for 1 min. At
this stage the sample still contains cellular debris.
3. Centrifuge for 15 min at 10,000 rpm (25°C), and collect
the supernatant in a 1.5-mL eppendorf tube.
4. Centrifuge again (15 min, 25°C), and transfer the su-
pernatant to a new eppendorf tube. Samples containing
solubilized proteins can then be stored at –80°C. Solu-
tions used to resuspend and solubilize proteins gener-
ally contain chaotropes, detergents, and reducing agents.
Chaotropes unfold proteins by breaking noncovalent

bonds. The most commonly used chaotropes are urea
and thiourea. They are often used in combination, which
improves protein solubilization. Detergents are need-
ed to improve protein solubilization in the presence of
chaotropic agents. CHAPS, a zwitter ionic detergent, has
solubilizing properties similar to those of Triton X-100
(a nonionic detergent) but has a more powerful effect in
preventing protein-protein interaction. SB3-10 is even
more efficient than CHAPS in solubilizing protein, but is
poorly soluble at high concentration in urea.
D. Preparation of Samples for IEF

1. Protein samples must be centrifuged once again before
use and the pellet discarded.
2. About 100 μg of total proteins are loaded per IEF gel (for
7-cm strip, follow supplier’s instructions).
3. Rehydrate the strip overnight.
4. Load the strip in IEF tray and run the optimised pro-
gram.
5. After IEF is complete, run the strip on SDS-PAGE (dis-
cussed in next chapter)
Precautions
1. The precipitation and rinsing solutions must be cold
when used. Always keep an acetone bottle at 4°C to pre-
pare these solutions.
2. Rehydration buffer contains high molarity of urea and
must not be heated above 30°C because isocyanate ions
are produced that would result in protein carbamylation.
3. A fine powder must be obtained for efficient protein ex-
traction. This may require precrushing of hard material
(e.g., mature maize grains). It is also possible to use an
automatic cryogenic crusher with a metallic ball (6 mm
diameter).
4. Up to pellet drying, it is important to work at a low tem-

perature (below 4°C) to limit protease action.
5. Several rinsing steps can profitably be applied with highly
pigmented samples, so that a white pellet is eventually
recovered. It is possible to extend rinsing overnight.
6. It is possible to store the dry pellet powder at –80°C be-
fore protein solubilization. However, in this case, it is bet-
ter to redry the powder before protein resolubilization.
References
1. B.D. Hames and D. Rickwood. Gel Electrophoresis of Pro-
teins: A Practical Approach 3rd Edition, The Practical Ap-
proach Series, Oxford University Press, 1998.
2. Ausubel, F.M., Brett, R., Kingston, R. E., Moore, D. D.,
Seidman, J.G., Smith, J.A., and Struhl, L. 1991. Current
Protocols in Molecular Biology, Vol. 1. Wiley. New York.
14.
Protein Quantification
Introduction
Proteins are biological macromolecules, occurring in all cells and
all parts of cells. Proteins also occur in great variety; thousands of
different kinds, ranging in size from relatively small peptides to
huge polymers with molecular weights in the millions. Moreover,
proteins exhibit enormous diversity of biological function and are
the most important final products of the information pathways.
Proteins are the molecular instruments through which genetic in-
formation is expressed.
Relatively simple monomeric subunits provide the key to
the structure of the thousands of different proteins. All proteins,
whether from the most ancient lines of bacteria or from the most
complex forms of life, are constructed from the same ubiquitous
set of 20 amino acids, covalently linked in characteristic linear
sequences. Because each of these amino acids has a side chain with
distinctive chemical properties, this group of 20 precursor mol-
ecules may be regarded as the alphabet in which the language of
protein structure is written.
What is the most remarkable is that cells can produce pro-
teins with strikingly different properties and activities by linking
the same 20 amino acids in many different combinations and se-
quences. From these building blocks different organisms can make
such widely diverse products as enzymes, hormones, antibodies,
transporters, muscle fibers, antibiotics and myriad other substanc-
es having distinct biological activities. Among these proteins, the
Protein Quantification 87
enzymes are the most varied and specialized. Virtually all cellular
reactions are catalyzed by enzymes. There are two methods for
quantitative estimation of proteins:
A. Protein Estimation By Lowry’s Method

Principle
The phenolic group of threonine, tyrosine and tyrptophan
residues in proteins produces a purple-blue coloured complex (of
sodium tungstate molybdate) on reaction with foline-ciocalteau
reagent having a maximum absorption peak at 660nm. Thus the
intensity of colour depends on the amount of these aromatic
amino acids present which varies for different proteins. Most
protein estimation techniques use bovine serum albumin (BSA)
as a standard protein, because of its low cost, high purity and easy
availability. This method is sensitive to about 10μg/ml protein
and is probably the most widely used protein assay. The incuba-
tion time is very critical for a reproducible assay. The reaction is
also dependent on pH 9 to 10.5.
Reagent Required
1. BSA stock solution (1mg/ml).
2. Analytical reagents
• Reagent A: 50ml of 2% Sodium carbonate (Na2CO3)
mixed with 50ml of 0.1N NaOH solution (0.4gm in
100ml D/W).
• Reagent B: 10ml of 1.56% Copper sulphate solution
(CuSO4) mixed with 10ml of 2.37% Sodium potassium
tartarate solution. Prepare analytical reagents by mixing
2ml of (B) with 100ml of (A).
3. Folin-Ciocalteau reagent: Dilute commercial reagent
with an equal volume of water immediately before use
(2ml of commercial reagent+2ml D/W).
4. Alkaline copper solution (Reagent C): Mix 50ml of A and
1ml of B prior to use.
5. Protein solution (Stock Standard).

• Weigh accurately 50mg of bovine serum albumin and
dissolve in double distilled water and make up final
volume to 50ml.
• Working standard: Dilute 10ml of the stock solution
to 50ml with distilled water in a standard flask. 1 ml
of this solution contains 200μg protein.
Procedure
1. Different dilution of BSA solution is prepared by mix-
ing stock BSA solution 1 mg/ml and DDW in clean test
tubes. The final volume in each test tube is 5ml. The BSA
range is 0.05 to 1mg/ml.
2. From these different dilutions pipette out 0.2ml protein
solution indifferent test tubes and add 2ml of alkaline
copper sulphate reagent. Mix well.
3. This solution is incubated at room temperature for 10
min.
4. Then add 0.2ml of folin-ciocalteau reagent to each tube
and incubate for 30 min. Zero the colorimeter with blank
and measure the optical density at 660nm.
5. Plot the standard curve of OD (y-axis) verses amount of
protein (x-axis)
6. Weigh 500mg of the test sample and grind well in a mor-
tar and pestle in 5-10 ml of phosphate buffer.
7. Centrifuge and use the supernatant for protein estima-
tion.
8. Pipette out 0.1 ml and 0.2ml of the sample extract in two
different test tubes respectively.
9. Make up the volume to 1ml in all the test tubes. A tube
with 1ml of water serves as the blank.
10. Add 5ml of reagent C to each tube including blank.
11. Mix well and allow standing for 10min.
12. Then add 0.5ml of Folin-Ciocalteau reagent, mix well
and incubate at room temperature for 30 min.
13. Record the OD at 660nm.

14. From the standard graph calculate the amount of protein
in the sample. Express the amount of protein in mg/gm
sample.
B. Protein Estimation By Bradford Method

Introduction
The Bradford assay is a fairly accurate, faster and a relatively
more sensitive in comparison to Lowry’s method. Bradford assay
is recommended for general use, especially for determining pro-
tein content of cell fractions and assessing protein concentrations
for gel electrophoresis. It is sensitive to about 5 to 200 micro-
grams protein, depending on the dye quality.
Principle
The assay is based on the observation that the absorbance
maximum for an acidic solution of coomassie brilliant blue
G-250 shifts from 465nm to 595nm after binding to a protein.
Both hydrophobic and ionic interactions stabilize the anionic
form of the dye causing a visible colour change.
Reagents
1. Bradford reagent: Dissolve 100 mg coomassie brilliant
blue G-250 in 50 ml 95% ethanol, add 100 ml 85%
(w/v) phosphoric acid. Dilute to 1 litre when the dye has
completely dissolved, filter through whatmann no. 1 pa-
per and store at room temperature.
2. The Bradford reagent should be a light brown in colour.
Filtration may have to be repeated to rid the reagent of
blue components.
Procedure
1. Prepare standards containing a range of 5 to 100 μg pro-
tein (BSA).
2. Cellular protein is isolated (as in Lowry’s method).

3. Add 5 ml dye reagent to the cell lysate and incubate 5 min.
4. Measure the absorbance at 595 nm.
Analysis
Prepare a standard curve of absorbance versus amount of protein
(mg/µl). Determine the amount of protein from test samples us-
ing the standard the curve.
C. SDS- PAGE
Principle
Sodium Dodecyl Sulphate Polyacrylamide Gel Eletropho-
resis (SDS-PAGE) is a technique widely used in biochemistry,
forensics, genetics, and molecular biology to separate proteins ac-
cording to their electrophoretic mobility (a function of length of
a polypeptide chain and its charge). SDS is an anionic detergent
which binds to polypeptide chain in a weight specific manner
i.e (1.4 gm SDS binds to 1 gm of protein) imparts net nega-
tive charge to a polypeptide; it also helps in lenearizing the pro-
teins by damaging the 3D structure due to repulsion in anonic
side chains of amino acid residues. In most proteins, the binding
of SDS to the polypeptide chain imparts an even distribution
of charge per unit mass. Thus, all protein complexes have same
charge density.
Proteins can be only separated on the basis of size (molecular
weight). Denaturation of protein mixture is done by heating at
1000C in presence of excess SDS with ß-mercaptoethanol. This
process cleaves the disulphide bonds in the proteins converting
them into single peptide. It is thus possible for an investigator to
determine the molecular weights of polypeptides on SDS-PAGE
by comparing relative mobilities against a set of standard refer-
ence protein of known molecular weights.
Reagents
1. 30% Acrylamide solution: Acrylamide 29 gm and N,
N’bisacrylamide 1 gm. Final volume is made up to 100ml

with distilled water, filtered and stored at 4oC.
2. Resolving gel buffer: (1.5M Tris-Cl buffer) 18.15gmTris
base is added to 50ml distilled water, pH is adjusted to
8.8 with 1N HCl and finally volume is made to 100ml
with distilled water.
3. 0.5 Tris-Cl buffer (pH 6.8): For this 3g of Tris-base is
added to 20ml distilled water, pH is adjusted to 6.8 with
1N HCl and final volume is made up to 50ml.
4. Electrophoresis buffer (pH 8.3): 3gmTris-base (0.025M),
14.42gm Glycine (1.192M) and 1gm SDS are dissolved
in 1L. DDW and pH adjusted to 8.3.
5. 10% w/v SDS: 1gm SDS in 10ml distilled water.
6. 10% Ammonium persulphate (APS): 0.01gm APS is dis-
solved in 0.1ml distilled water. Freshly prepared.
7. Tetraethyl Methylene Diamine (TEMED) sample buffer:
Prepared by adding 3.8ml DDW, 0.5ml Tris-Cl buffer
of pH 6.8, 0.8ml glycerol, 1.6ml 10% SDS, 0.4ml ß-
Mercaptoethanol, 0.4ml (1.5% w/v) bromophenol blue.
8. Staining Solution: 90ml methanol, 20ml acetic acid add-
ed to 90ml DDW. 0.1gm coomassie brilliant blue R-250
added to it and filtered through whatmann no. 1 filter
paper.
9. Destaining solution: It is prepared by adding 50ml meth-
anol, 100ml acetic acid to 850ml DDW.
Procedure
1. Clean and wipe the glass plates and spacers with alcohol
and dry.
2. Plates are then assembled along with spacer.
3. Comb is placed in between the two plates and clamps are
set appropriately on the right, left and on the bottom side
of the plate (alternatively the plates can be sealed with
steelgrip tapes).
4. Plates are leveled on a flat surface.

5. Pour the resolving gel mixture between the plates and in-
sert the comb.
6. Leave for 20-30 minutes for polymerization.
7. Comb is removed slowly and wells are rinsed properly
with distilled water, decant excess water. Clamps are re-
moved.
8. It is then put into electrophoretic system.
9. Pour the electophoretic buffer into lower buffer chamber
and the gel system is put in to it by slightly tilting it to
avoid air bubbles.
10. Pour the running buffer in the upper buffer chamber.
11. Load the samples into wells and then place the lid on top.
Power cord is attached for power supply; care should be
taken that anode is connected with anode and cathode
with cathode.
12. Apply 100-200 V and run the gel till the dye front comes
to the bottom of the gel.
13. After the electrophoresis is complete, power supply is
turned off and glass plates are gently removed.
14. Gently remove the spacers and gel from the plate, wash it
with distilled water and cut the corner from the bottom
of the gel that is close to well 1.
15. Gel is stained in a tray for 8-10 hours, removed and keep
in destaining solution till background gets clear.
Sample Preparation
Samples may be any culture, tissue or forensic sample, for
example prokaryotic or eukaryotic cells, tissues, viruses, environ-
mental samples, or purified proteins. In the case of solid tissues,
these are often first lysed mechanically using a blender (for larger
sample volumes), using a homogenizer (smaller volumes), by us-
ing cycling of high pressure. Cells may also be lysed by one of
the above mechanical methods. In the case of tissues or cells, a
combination of biochemical and mechanical techniques includ-

ing various types of filtration and centrifugation may be used
to separate different cell compartments and organelles prior to
electrophoresis.
The sample to be analyzed is mixed with SDS, an anionic
detergent which denatures secondary and non-disulfide-linked
tertiary structures, and applies a negative charge to each protein
in proportion to its mass. Heating the samples to at least 60oC
further promotes protein denaturation, helping SDS to bind. A
tracking dye may be added to the protein solution. This typically
has higher electrophoresis mobility than the proteins to allow the
experimenter to track the progess of the protein solution through
the gel during electrophoresis run.
Preparing Polyacrylamide Gel

The gels consist of acrylamide, bisacrylamide, SDS and a
buffer with an adjusted pH. The solution may be degassed under
a vacuum to prevent the formation of air bubbles during polym-
erization. Alternatively, butanol may be added to the resolving
gel after it is poured, as butane removes bubbles and makes the
surface smooth. A source of free radicals and a stabilizer such as
ammonium persulfate and TEMED are added to initiate polym-
erization. The polymerization reaction result in a gel because of
the added bisacrylamide, generally about 1 part in 35 relative to
acrylamide, which can form cross-links between two polyacrla-
mide molecules. The ratio of acrylamide to bisacrylamide can
be varied for different purposes. The acrylamide concentration
of the gel can also be varied, generally in the range from 5% to
25%. Lower percentage gels are better for resolving very high
molecular weight proteins, while much higher percentages are
needed to resolve smaller proteins.
Gels are usually polymerized between two glass plates (gel
caster), with comb inserted at the top to create the sample wells.
After the gel is polymerized the comb is removed and the gel is
ready for electrophoresis.
Electrophoresis
Various buffer systems are used in SDS-PAGE depending on
the nature of the sample and the experimental objective. When an
electric field is applied on the gel, causing the negatively-charged
proteins to migrate across the gel towards the positive (anode)
electrode. Depending on their size, each protein will move dif-
ferently through the gel matrix: short proteins will move more
easily through the pores in the gel, while larger ones will have
more difficulty (they encounter more resistance). After certain
period (usually a few hours, through this depends on the voltage
applied across the gel; protein migration occurs more quickly at
higher voltages) the proteins will have differentially migrated in
the gel based on their size; smaller proteins will have travelled far-
ther down the gel, while larger ones will have remained closer to
the point of origin. Proteins may therefore be separated roughly
according to size; however certain glycoproteins behave anoma-
lously on SDS gels.
SDS-PAGE is usually the first choice as an assay of protein
purity due to its reliability and ease. The presence of SDS and the
denaturing step causes proteins to be separated approximately
based on size, but aberrant migration of some proteins may oc-
cur. Different proteins may also stain differently, which inter-
feres with quantification by staining. PAGE may also be used
as a preparative technique for protein purification. For example,
quantitative preparative native continuous polyacrylamide gel
electrophoresis (QPNC-PAGE) is a method for separating native
metalloproteins in complex biological matrices.
Role of Chemical Ingredients

Polyacrylamide gel
Possesses several electrophoretically desirable features that
make it a versatile medium. It is a synthetic, thermo-stable, trans-
parent, strong, chemically inert gel, and can be prepared with a
wide range of average pore sizes. The pore size of a gel is deter-
mined by two factors, the total amount of acrylamide present
(%T) (T= Total concentration of acrylamide and bisacrylamide

monomer) and the amount of cross-linker (%C) (C= bisacryla-
mide concentration). Pore size decreases with increasing %T; with
cross-linking, 5%C gives the smallest pore size. Any increases or
decrease in %C from 5% increases the pore size, as pore size with
respect to %C is parabolic function with vertex as 5%C. This
appears to be because of non-homogeneous bundling of polymer
strands within the gel. This gel material can also withstand high
voltage gradients, is amenable to various staining and destaining
procedures, and can be digested to extract separated fractions or
dried for autoradiography and permanent recording.
Chemical buffer
Buffer Stabilizes the pH to the desired value within the gel it-
self and in the electrophoresis buffer. The choice of buffer also af-
fects the electrophoretic mobility of the buffer counter-ions and
thereby the resolution of the gel. The buffer should also be unre-
active and not modify or react with proteins. Common buffers
in SDS-PAGE include Tris, Bis-Tris, or imidazole. Counter-ion
balances the intrinsic charge of the buffer ion and also affects the
electric field strength during electrophoresis. Highly charged and
mobile ions are often avoided in SDS-PAGE buffers, but may be
included in the gel itself, where it migrates ahead of the protein.
Most popular counter-ion is glycine tricine. Glycine has been
used as the source of trailing ion or slow ion because its pKa is
9.69 and mobility of glycinate are such that the effective mobility
can be set a value below that of the slowest known proteins of net
negative charge in the pH range. The minimum pH of this range
is approximately 8.0.
Acrylamide (C3H5NO; MW: 71.08)

When dissolved in water slow and spontaneous auto-poly-
merisation of acrylamide takes place joining molecules together
in head to tail fashion to form long single-chain polymers. The
presence of a free radical-generating system greatly accelerates
polymerization. This kind of reaction is known as vinyl addi-
tion polymerisation. A solution of these polymer chains becomes

viscous but does not form a gel, because the chain simply slides
over one another. Gel formation requires linking various chains
together. Acrylamide is a neurotoxin. It is also essential to store
acrylamide in a cool, dark and dry place to reduce auto-polymer-
isation and hydrolysis.
Bisacrylamide (N, N’-Methylenebisacrylamide)

(C7H10O2; MW: 154.17)
Bisacrylamide is the most frequently used cross linking
agent for polyacrylamide gels. Chemically it can be thought of
as two acrylamide molecules coupled at their non-reactive ends.
Bisacrylamide can crosslink two polyacrylamide chains to one
another, thereby resulting in a gel.
Sodium Dodecyl Sulfate (SDS)

(C12H25NaO4S; MW: 288.38)
SDS is an anionic detergent used to denature native proteins
to unfold, individual polypeptides. When a protein mixture is
heated to 100oC in presence of SDS, the detergent wraps around
the polypeptide backbone. It binds to polypeptides in a constant
mass ratio of 1.4g SDS/gm of polypeptide. In this process, the in-
trinsic charge of polypeptide becomes negligible when compared
to the negative charge contributed by SDS. Thus polypeptides
after treatment with SDS becomes rod-like, linear structures pos-
sessing a uniform charge density i.e. same net negative charge per
unit length. The electrophortic mobility of these proteins will be
a linear function of the logarithm of their molecular weight.
Without SDS, different proteins with similar molecular
weights would migrate differently in mass-charge ratio, as each
protein has an isoelectric point and molecular weight particular
to its primary structure. This is known as native PAGE. Adding
SDS solves this problem, as it binds to and unfolds the protein,
giving a uniform net negative charge to the polypeptide.
Ammonium persulfate
(APS) (N2H8S2O8; MW: 228.2)
APS is a source of free radicals and is often used as an initia-
tor for gel polymerisation. An alternative source of free radicals
is riboflavin, which generated free radicals in a photochemical
reaction.
TEMED (N, N, N’, N’-tetramethylienediamine)

(C6H16N2; MW: 116.21)
TEMED stabilizes free radicals and improves polymeriza-
tion. The rate of polymerisation and properties of the resulting
gel depend on the concentrations of free radicals. Increasing the
amount of free radical results in a decrease in the average poly-
mer chain length and an increase in gel turbidity.
Chemicals for processing and visualization

The following chemicals are used for processing of gel and
the protein samples visualized in it:
Tracking dye
As proteins are mostly colourless, their progress through the
gel during electrophoresis cannot be easily followed. Anionic
dyes of a known electrophoretic mobility are therefore usually in-
cluded in the SDS-PAGE sample buffer. A very common track-
ing dye is bromophenol blue (3’, 3’’, 5’, 5’’ tetra bromo phenol
sulfo nphthalein). This dye is coloured at alkaline and neutral
pH and is also negatively charged and moves toward anode. Be-
ing a highly mobile molecule (due to its low molecular weight)
it moves ahead of most proteins. As it reaches the anode power
pack is turned off. It can weakly bind to some proteins and im-
part a blue colour.
Loading aids
Most SDS-PAGE systems are loaded from the top into wells
within the gel. To ensure that the sample sinks to the bottom of
the gel, sample buffer is supplemented with additives that in-
crease the density of the sample these additives should be non-
ionic and non-reactive with proteins to avoid interfering with
electrophoresis. Common additives are glycerol and sucrose.
Coomassie Brilliant Blue R-250

(C45H44N3NaO7S2; MW: 825.97)
CBB is one of the most popular protein stains. It is an ani-
onic dye, which non-specifically binds to proteins. The structure
of CBB is predominantly non-polar, and it is usually used in
methanolic solution acidified with acetic acid. Proteins in the gel
are fixed by acetic acid and simultaneously stained. The excess
dye incorporated into the gel can be removed by destaining with
the same solution without the dye. The proteins are detected as
blue bands on a clear background. As SDS is also anionic, it may
interfere with staining process. Therefore, large volume of stain-
ing solution is recommended, at least ten times the volume of
the gel.
Precautions
1. The glass plates should be absolutely clean.
2. No air bubbles should be there inside the glass plates.
3. Separate pipettes should be used to avoid contamination.
4. Care should be taken while handling acrylamide since
unpolymerized acrylamide is neurotoxic.
SYMPTOMS POSSIBLE CAUSES REMEDY

Distortion of 2D Pattern
Vertical gel format Degas the gel solution.
Uneven polymeri- Polymerization can be ac-
zation of gel due to celerated by increasing by
incomplete polym- 50% the amount of am-
erization, too rapid monium persulfate and
polymerization, or TEMED used. Polym-
leakage during gel erization can be slowed
casting. by decreasing by 33% the
amount of ammonium
persulfate and TEMED
used.
Ensure that there is no
leakage during gel casting.
Horizontal Streaking or Incompletely Focused Spots
Sample not complete- Make sure that the sample
ly solubilized prior to is completely and stably
application. solubilized. Repeated pre-
cipitation-resolubilization
cycles produce or increase
horizontal streaking.
Sample is poorly solu- Increase the concentration
ble in rehydration so- of the solubilizing compo-
lution. nents in the rehydration
solution.
Increase concentration of
IPG buffer
Interfering substrates. Modify sample prepara-

Nonprotein impuri- tion to limit these con-
ties in the sample can taminants.
interfere with IEF,
causing horizontal
streaking in the final
2D result, particular-
ly toward the acidic
side of the gel.
Ionic impurities in Reduce salt concentration

sample. to below 10 mm by dilu-
tion or desalt the sample
by dialysis. Precipitation
with TCA and acetone
and subsequent resuspen-
sion is another effective
desalting technique that
removes lipids, nucleo-
tides, and other small mol-
ecules. Specific and non-
specific losses of proteins
can occur with dialysis, gel
chromatography, and pre-
cipitation/resuspension of
samples.
If the sample cannot be
modified, reduce the ef-
fect of ionic impurities by
modifying the IEF proto-
col. Limit the voltage to
100–150 V for 2 hours,
then resume a normal
voltage step program. This
pre-step allows the ions in
the sample to move to the
ends of the IPG strip.
Ionic detergent in If the ionic detergent SDS
sample. is used in sample prepa-
ration, the final concen-
tration must not exceed
0.25% after dilution into
the rehydration solution.
Additionally, the concen-
tration of the nonionic
detergent present must be
at least 8 times higher than
the concentration of any
ionic detergent to ensure
complete removal of SDS
from the proteins.
High sample load. Load less sample.

Micropreparative separa-
tions require clean sample.
Modify sample prepara-
tion to limit concentra-
tions.
Program a low initial volt-
age gradually. Extend fo-
cusing time.
Underfocusing. Fo- Prolong focusing time.
cusing time was
not long enough to
achieve steadystate
focusing.
Overfocusing. Ex- Reduce focusing time.
tended focusing times
(>100,000 Vh) may
result in electroen-
dosmotic water and
protein movement,
which can produce
horizontal smearing.
Vertical Streaking
Insufficient equilibra- Prolong equilibration
tion. time.
Se c o n d d i m e n s i o n Prepare fresh solutions.
buffer solutions pre- Use 0.1% (w/v) SDS.
pared incorrectly.
Insufficient SDS in
SDS electrophoresis
buffer.
Vertical Gap in 2D Pattern
Impurities in sample. Modify sample prepara-
tion.
Impurities in rehy- Use only high-quality rea-

dration solution com- gents.
ponents. Deionize urea solutions.
Bubble between IPG Ensure that no bubbles are

strip and top surface trapped between IPG strip
of second-dimension and the top surface of the
gel. second-dimension gel.
Vertical Regions of Poor focusing
The IPG strip was not Ensure that the IPG strips
fully rehydrated. are rehydrated with suffi-
cient volume of rehydra-
tion solution.
Remove any large bubbles
trapped under the IPG
strip after rehydration so-
lution is applied.
Check that the rehydra-
tion solution is evenly
spread along the entire
length of the IPG strip.
Properly wash glass plates.
Scavenge any excess or re-
sidual thiol reducing agent
with iodoacetamide before
loading the IPG strips
onto the second-dimen-
sion gel.
Adapted from: www.biorad.com
References
1. Anderson NL and Anderson NG. 1998. Proteome and prot-
eomics: new technologies, new concepts, and new words. Elec-
trophoresis 19 (11): 1853–61.
2. Blackstock WP and Weir MP. 1999. Proteomics: quantita-
tive and physical mapping of cellular proteins. Trends in Bio-
technology 17 (3): 121–7.
3. Wilkins MR, et al. 1996. From Proteins to Proteomes: Large
Scale Protein Identification by Two-Dimensional Elec-
trophoresis and Amino Acid Analysis. Nature Biotechnol-
ogy 14 (1): 61–65.
Western Blotting 103
15.
Western Blotting
Principle
Specific proteins separated by resolving gel electrophoresis in a
complex mixture are transferred on to the nitrocellulose mem-
brane and detected by the use of labelled antibodies. Two differ-
ent antibodies are used one (primary antibody) specific for the
desired protein and the other (secondary antibody) linked to a
reporter enzyme (horse radish peroxidase, HRP). HRP converts
the substrate H2O2 into water and molecular oxygen, which in
turn oxidize 3,3’-diaminobenzidine (DAB) to from an intense
brown insoluble residue indicating the presence of particular
protein on the nitrocellulose paper.
Reagents
1. 30% Acrylamide solution: Acrylamide 29 gm and N, N’
bisacrylamide 1 gm. Final volume is made up to 100ml
with DDW, filtered and stored at 4oC.
2. Resolving gel buffer: (1.5M Tris-Cl buffer) 18.15gm
Tris-base is added to 50ml DDW, pH is adjusted to 8.8
with 1N HCl and finally volume is made to 100ml with
DDW.
3. 0.5 Tris-Cl buffer (pH 6.8): For this 3gm of Tris-base is
added to 20ml DDW, pH is adjusted to 6.8 with 1N HCl
and final volume is made up to 50ml.
4. Electrophoresis buffer (pH 8.3): 3gm Tris-base
(0.025M), 14.42gm Glycine (1.192M) and 1gm SDS are
dissolved in 1L. DDW and pH adjusted to 8.3

5. 10% w/v SDS: 1gm SDS in 10ml DDW.
6. 10% Ammonium persulphate (APS): 0.01gm APS is
dissolved in 0.1ml DDW. Always use freshly prepared.
7. Tetraethyl Methylene Diamine (TEMED) sample
buffer: Prepared by adding 3.8ml DDW, 0.5ml Tris-Cl
buffer of pH 6.8, 0.8 ml glycerol, 1.6ml 10% SDS, 0.4ml
ß-mercaptoethanol, 0.4 ml (1.5 w/v) bromophenol blue.
8. Staining Solution: 90ml methanol, 20ml acetic acid
added to 90ml DDW. 0.1gm CBB R-250 added to it and
filtered through whatmann no.1 filter paper.
9. Destaining solution: It is prepared by adding 100 ml
methanol, 50 ml glacial acetic acid to 850ml DDW.
10. 50mM Phosphate buffer (pH 7.4): Dissolved 7.12gm of
Na2HPO4 and 1.56gm of NaH2PO4 in 100ml of DDW.
11. Transfer buffer (pH 8.3): Dissolve 1.513gm of Tris base
(25mM) and 7.206gm Glycine in 400 ml of DDW pH
was adjusted to 8.3 with 1N HCl and the final volume
was made up to 500ml.
12. PBST: Phosphate buffered saline with Tween-20.
13. Washing solution (6mM PBS pH 7.4): Add 0.4272gm
of Na2HPO4, 0.0936gm of NaH2PO4, 3.973gm NaCl
and 0.1gm KCl in 500 ml DDW.
14. Antibody dilution solution: Take 50ml of PBS and add
one drop of 0.1% of tween-20, mix gently.
15. Blocking solution: Add 2.5gm non-fat dry milk powder
to 50ml of PBST.
16. Primary and secondary antibodies.
17. Nitrocellulose membrane and blotting paper.
18. Semidry transfer unit.
Procedure
1. Clean and wipe the glass plates and spacers with alcohol
and air dry.
2. Plates are then assembled along with spacer.

3. Comb is placed in between the two plates and clamps are
set appropriately on the right, left and on the bottom side
of the plate.
4. Plates are levelled on a flat surface and sides are sealed by
tape/agar.
5. Pour the resolving gel mixture between the plates and fix
the comb.
6. Leave 20-30 minutes for polymerization.
7. Comb is removed slowly and wells are rinsed properly
with DDW, decant excess water. Clamps are then re-
moved.
8. Gel casted plates are then put into electrophoretic system.
9. Pour the running buffer in electrophoretic tank and the
gel system is put into it by slightly tilting it to avoid air
bubbles.
10. Pour the electrode buffer in the upper buffer chamber.
11. Load the prestained molecular weight marker in the first
lane.
12. Load the samples into wells and then place the lid on top.
Power cord is attached for power supply taking care of its
polarity i.e. Anode is connected with anode and cathode
with cathode.
13. Apply 100-200 V and run the gel till the dye front comes
to the bottom of the gel.
14. Run the gel till the dye reaches approximately at the bot-
tom of gel.
15. After electrophoresis is complete, power supply is turned
off and glass plates are gently removed.
16. Gently remove the spacers and gel from the plate, wash it
with distilled water and cut the corner from the bottom
of the gel that is close to first well.
17. Gel is stained in a tray overnight, removed and kept in
destaining solution till background gets clear.
Western transfer
1. Keep the gel in transfer buffer for 30 min.
2. Cut 6-8 sheets of absorbent paper (whatmann 3mm) and
a sheet of nitrocellulose membrane 1mm less in size to
that of gel.
3. Soak the blotting paper and nitrocellulose membrane in
the transfer buffer for 30 min.
4. Prepare the transfer stack in following manner: Anode
base plate-blotting paper-nitrocellulose membrane-gel-
blotting paper-upper lid cathode.
5. Connect the transfer set with relevant electrodes of the
power supply and allow transferring for 1 hour at 0.8mA/
cm2.
6. After the transfer, carefully dissemble the stack and mark
the nitrocellulose membrane to follow the orientation.
Immunodectection
1. Rinse the membrane in PBS and keep it in block solution
for 1 hr at room temperature on a shaker.
2. Incubate the membrane in primary antibody (catalase:
dilute 1000 times in PBST with 5% skimmed milk solu-
tion) for 1 hr at room temperature with gentle shaking.
3. Wash the membrane 3 times for 5 min each in PBS and
add secondary antibody (HRP conjugated antibody; di-
lute 1:2000 in PBST with 2.5% skimmed milk). Incu-
bate for 1 hour at room temperature with shaking.
4. Wash the membrane in PBS 3-4 times for 5 min each.
5. Detect the antibody (catalase) expression by putting the
membrane in developer (10ml of 0.01% DAB) with 10μl
of H2O2 for 1 min observe the membrane for result.
Tranfer buffer
Cathod(–)
Filter
paper
Gel
Nitrocellulose
membrane
Filter
paper
Anode(+) Membrane
(with transferred
proteins)
Fig. Gel setting in a western blot
References
1. Bolt and Mahoney. 1997. High-efficiency blotting of pro-
teins of diverse sizes following sodium dodecyl sulfate–poly-
acrylamide, gel electrophoresis. Analytical Biochemistry 247,
185–192.
2. Harlow, Ed and Lane D. 1999. Using Antibodies. Cold
Spring Harbor, New York: Cold Spring Harbor Laboratory
Press.
3. Hames BD and Rickwood D. 1998 . Gel Electrophoresis of
Proteins: A Practical Approach 3rd Edition, the Practical
Approach Series, Oxford University Press.
Commonly Used Biochemical Data

Nucleic Acid Data
Average weight of a DNA base pair (sodium salt) = 650 daltons
1.0 A260 unit ds DNA = 50 μg/ml = 0.15 mM (in nucleotides)
1.0 A260 unit ss DNA = 33 μg/ml = 0.10 mM (in nucleotides)
1.0 A260 unit ss RNA = 33 μg/ml = 0.10 mM (in nucleotides)
MW of a ds DNA molecule = (# of base pairs) × (650 daltons/
base pair)
1 μg of 1000 bp DNA = 1.52 pmol = 9.1 × 1011 molecules
1 μg of pBR322 DNA = 0.35 pmol = 2.1 × 1011 molecules
1 μg of l DNA = 0.03 pmol = 1.8 X 1010 molecules
Agarose gel resolution

% Gel Optimum Resolution for linear DNA (kb)
0.5 30 to 1.0
0.7 12 to 0.8
1.0 10 to 0.5
1.2 7 to 0.4
1.5 3 to 0.2
Polyacrylamide gel resolution

% Gel Optimum Resolution for linear DNA (pb)
3.5 1000-2000
5.0 80-500
8.0 60-400
12.0 40-200
15.0 25-150
20.0 6-100
109
Linear Range of Separation for protein

(SDS-PAGE)
% Gel Protein M.W. (kD)
15 10-43
12 12-60
10 20-80
7.5 36-94
5.0 57-212
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LAB MANUAL ON MOLECULAR BIOLOGY

Book · January 2016

Manual for Laboratory View project

The user has requested enhancement of the downloaded file.

1. General Laboratory Procedures, Equipment Use and

These chemicals are not harmful if used properly: always

II. Preparation of Solutions

A molar solution is one in which 1 litre of solution contains

chemical. Weigh out the desired amount of chemical(s).

D. Glassware and Plastic Ware

of polypropylene are turbid and are resistant to many chemicals,

III. Disposal of Buffers and Chemicals

C. Autoclave Operating Procedures

1. Make sure chamber pressure is zero before opening the

Proper Use of the Microscope

the centre of the field of view and re-adjust the mirror,

Working with DNA

conditions for storing DNA. –200C causes extensive single and

ture tube. Sufficient numbers of bacteria are present in

date and your initials. Immediately place into a dry ice/ethanol

Instructions for maintaining lab book

B. Background information: This section should include

D. Procedure/ Method: Write down exactly what you are

in low yield; therefore, the culture should be in the log

B. Nucleic acid purification

C. Nucleic acid precipitation

2. Precipitate the DNA by centrifugation at 14,000 rpm for

D. Nucleic acid resuspension and final cleanup

molecular-weight (>10 kb) or low-copy-number plasmids than

2. Grow with shaking at 37°C overnight.

Nicked Uncut Cut

enzymes. This can be overcome by washing the cell pellet

tissue may be dried or fresh. Scrape clean or cut away surface

slowly adding 6N HCl and make up the volume of solu-

9. Sodium acetate (3 M, pH 8.0): Dissolve 24.6 g of sodium

Collection Of Plant Materials

8. Transfer the top aqueous phase to a new 15 ml centrifuge

trifuge at 10,000 rpm for 5 minutes at room tempera-

6. Sodium acetate (3 M, pH 8.0): Dissolve 24.6gm of so-

14. Decant the supernatant and the DNA pellet is left at

A. Estimation of DNA by Nanodrop

Often DNA isolated with a commercial kit shows

B. Estimation of DNA by Diphenylamine

3. Make up the volume to 1 ml in all the test tubes. A tube

5. Let the solution cool down to room temperature then add

(1) (2) (3)

(4) (5) (6)

Fig. Agarose Gel Electrophoresis.

producing the necessary single-stranded DNA template for the

Denature and anneal

Denature and anneal

Fig: PCR Amplification

Extension of the primer by the thermostable polymerase lasts

allow easy cloning into “T”-vectors (pGEM-T) which possess

applications. For example, restriction enzyme sites can be placed

ally by the addition of the critical component when the reaction

Nucleic Acid Cross-Contamination

vortex the tube for 10 seconds and briefly centrifuge in

Incorrect primer Verify that the primers are complemen-

system may be produced by the addition of more salt, in which

the phenol at -20°C.

B. Lysis and Extraction with PEG 6000

B. Estimation of DNA by Diphenylamine

(a) A blob at the running edge end of the gel—the RNA