Laboratory Manual of Basic Molecular Bio

Laboratory Manual of
Basic Molecular Biology

Techniques
Professor Dr. Najat Abdulrazzaq Hasan

(MbChB, MSc, PhD in clinical Biochemistry)
DEDICATION
In Loving Memory of my Father, Mother and Husband

Samir Khudhur
Who devoted their lives in supporting me and all my brothers and sisters
to establish higher degree of scientific levels and without them, this book
would not exist.
To my beloved son Aws and his family
Who have made me stronger, better and more fulfilled than I could have
ever imagined and inspired me to prepare this teaching laboratory manual.
For their endless support, encouragement during writing and archiving it.
Najat A. Hasan
2
PREFACE
Science may be the construction of rules and relationships that define a world
that matches our world as closely as possible. Science involves learning, testing
ideas, information collection and analysis. Scientists are explorers who go
beyond what it is currently known.
The purpose of this laboratory manual is to introduce undergraduate and

graduate students from different discipline to basic techniques used in
Biochemistry and Molecular Biology Laboratories and ensure that they can
understand the theoretical aspects of molecular biology and to master the lab
skills necessary to be competitive in their scientific life and researches. We
present a collection of fourteen experiments that teach students knowledge about
Molecular Biology laboratory hazards, sterile techniques, accurate pipetting, and
micro centrifuge usage, DNA extraction from different living sources, RNA
purification, quantification, and electrophoresis and purity assessment of the
extracted nucleic acids. Base composition of RNA and DNA and how it affect the
topology of DNA during DNA melting and annealing biological process and the
specificity of the restriction enzyme digestion will be delineated.
This laboratory manual takes the advantage of the fact that a biochemistry
lecture course is pre- or co-requisite to taking a biochemistry laboratory course.
As a result students are expected to be familiar with the general principles behind
each experiment from a lecture course.
Two-to-three students per team works well to maximize peer interaction

while still making sure that each student has a chance to intellectually contribute
to the assignments. The course was typically taught by a Molecular Biologist and
trained Biochemist in Molecular Biology and teaching assistants. Pre-lab
questions are designed to focus students’ attention to the most important points
in the experiment. They will be asked to complete post-lab assignment before
coming to next laboratory. Some problem sets can be answered during
laboratory to fill the waiting time during Molecular Biology experiments.
Dr. Najat A. Hasan, 2019

TABLE OF CONTENTS
Seq. Title Page
Laboratory session No. 1: General Laboratory Procedures
1. 6
and Safety Considerations
Laboratory session No.2: Introduction to DNA Extractions,
2. 10
Preparation of crude DNA extracts from different sources
Laboratory session No. 3: Phenol/ chloroform Extraction of
3. 18
DNA
Laboratory session No. 4: Determination of the Purity and
4. 22
Quantity of DNA
Laboratory session No.5: Determination of DNA Melting
5. 25
Point and GC content
Laboratory session No.6: Agarose Gel Electrophoresis of
6. 32
DNA
7. Laboratory session No. 7: Principle of the PCR 38
Laboratory session No. 8: Annealing Temperature and
8. 46
Primer Design
9. Laboratory session No. 9: Preparation of PCR Master Mix 50
Laboratory session No. 10: Spin column method of RNA
10. isolation by acid 54
guanidinium thiocyanate–phenol–Chloroform extraction
Laboratory session No.11: Analyzing RNA Quality and
11. 59
integrity
Laboratory session No.12: Estimation of RNA by Orcinol
12. 66
Reaction
Laboratory session No.13: Nucleotide Composition of
13. 68
RNA
Laboratory session No.14: Restriction Enzyme digestion
14. 71
of DNA molecule –DNA fingerprints
15. References 80
Appendix A:
16. Safety rules and regulations in Laboratory of molecular 83
biology
Disposal and
17. 84
Decontamination of Ethidium Bromide
18. Appendix C: Microcentrifuges and centrifugation 91
4
Laboratory session No. 1
General Laboratory Procedures and Safety

Considerations
I. Safety Procedures
A. Chemicals
All manufacturers of hazardous materials are required by law to supply the user
with the information on any hazards associated with their chemicals. This
information is supplied in the form of Material Safety Data Sheets (MSDS)
which contains the chemical name, the chemical abstract service-CAS#, health
hazard data, including first aid treatment, physical data, fire and explosion hazard
data, reactivity data, spill or leak procedures, and any special precautions
needed when handling that chemical. A file containing MSDS information on the
hazardous substances should be kept in the lab.
The following chemicals are particularly noteworthy in molecular biology labs:
 Phenol - can cause severe burns
 Ethidium bromide - carcinogen
 Acrylamide - potential neurotoxin
These chemicals are not harmful if used properly such as wear gloves avoid
mouth-pipetting them. In case of an accidental splash any of these chemicals on
the skin, immediately rinse the area thoroughly with water and inform the
instructor.
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 exposure. 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 clean. All solutions and everything stored in an
incubator, refrigerator, etc. must be carefully labeled. In order to limit confusion,
each person should use his initials or other unique designation for labeling
plates, etc. Unlabeled material found in the refrigerators, incubators, or freezers
may be destroyed.
II. Preparation of Solutions
A. Calculation of Molar, % and "X" Solutions.
1. . A molar solution is one in which 1 liter of solution contains the number
of grams equal to its molecular weight. Ex. To make up 100 ml of a 5M
NaCl solution = 58.456 (mw of NaCl) g/mol x 5 moles/liter x 0.1 liter (100
ml) = 29.29 g in 100 ml of solution
2. Percent solutions. Percentage (w/v) = weight (g) in 100 ml of solution;
Percentage (v/v) = volume (ml) in 100 ml of solution. Ex. To make a 1%
solution of agarose in Tris borate EDTA (TBE) buffer, weight 1g 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
working solution) and are then diluted such that the final concentration of
the buffer in the reaction is 1X.
B. Steps in Solution Preparation:
6
1. After preparation of the particular solution and the bottle is labeled and
autoclave at 121° C for 20 minutes. Some solutions cannot be autoclaved, for
example, sodium dodecyl sulfate (SDS). These should be filter sterilized through
a 0.22 μ m or 0.45 μ m Millipore filter.
The agarose can be melted in a microwave.
2. Concentrated solutions, e.g. 1M Tris-HCl pH=8.0, 5M NaCl, can be used to
make working stocks by adding the appropriate amount of the autoclaved
double-distilled water in a sterile vessel.
C. Glassware’s and Plastic Wares.
Glass and plastic wares used for molecular biology must be very 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 at 121° C or
baked at 150 degrees C for 1 hour. For experiments with RNA, glassware and
solutions are treated with diethyl-pyro carbonate (DEPEC) to inhibit RNases
which can be resistant to autoclaving. Micro pipet tips and microfuge tubes
should be autoclaved before use or preferably use filter tips.
III. Disposal of Buffers and Chemicals

1. Any uncontaminated, solidified agar or agarose should be discarded in the
trash, not in the sink, and the bottles rinsed well.
2. Organic reagents, e.g. phenol, should be used in a fume hood and all
organic waste should be disposed of in a labeled container, not in the trash or the
sink.
4. Ethidium bromide is a mutagenic substance that should be treated before
disposal and should be handled only with gloves. Ethidium bromide should be
disposed of in a labeled container.
5. Dirty glassware should be washed with hot soapy water, rinsed well with
hot water, and rinsed three times with distilled water.
IV. Equipment
It is to everyone's advantage to keep the equipment in good working condition.
Report any malfunction immediately. If the supply is running low, please notify
either the instructor/lab manager before the supply is completely exhausted.
A. Micropipettors
Ensure that all pipettes are accurate and are maintained in good working order.
.DO NOT DROP IT ON THE FLOOR.
B. Operating Instructions for Spectrophotometer:

To measure the absorbance of a solution in the short-wave range (<300 nm)
use the quartz cuvettes. Disposable plastic cuvettes are available for reading in
the visible range.
V. Working with DNA
A. Storage
The following properties of reagents and conditions are of important
considerations in processing and storing DNA and RNA.
1. Heavy metals promote phosphodiester breakage.
2. EDTA is an excellent heavy metal chelator.
3. Free radicals are formed from chemical breakdown and radiation and they
cause phosphodiester breakage. UV light at 260 nm causes a variety of
lesions, including thymine dimers and cross-link between two DNA
strands.
4. Ethidium bromide causes photo oxidation of DNA and breakage of DNA
phosphodiester bonds.
5. Nucleases are found on human skin; therefore, avoid direct or indirect
contact between nucleic acids and fingers. Most DNases are not very
stable; however, many RNases are very stable and can adsorb to glass or
8
plastic and remain active. The 5 ° C is one of the best and simplest
conditions for storing DNA for short period. Whereas, -20 °C causes single
and double strand breaks when used for few months storage. The -70 ° C
is 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. There is about one phosphodiester break per 200 kb of DNA per year.
VI. Sterile Technique
1. All liquid media and Glass wares must be autoclaved immediately after it
is prepared.
2. Always use a fresh solutions, sterile gloves, plastic wares, pipette or pipette
tip when pipetting culture media.
Laboratory session No.2
Introduction to DNA Extractions, Preparation of crude

DNA extracts from different sources
Onion DNA Extraction
Protocol
1. Cut an inch square out of the center of 3 medium onions. Chop and place
in a blender.
2. Add 100 ml of detergent/salt solution.
3. Blend on high 30 sec-1 minute.
4. Strain the mixture into a beaker using a strainer with a coffee filter.
5. Add 20-30 ml meat tenderizer and stir to mix.
6. Place 2 ml filtrate in a test tube.
7. Pour 2 ml ice cold ethanol carefully down the side of the tube to form a
layer.
8. Let the mixture sit undisturbed 2-3 minutes until bubbling stops.
9. The DNA will float in the alcohol. Swirl a glass stirring rod at the interface
of the two layers to see the small threads of DNA.
10
Wheat Germ DNA Extraction
Protocol
1. Add 100 ml distilled water to a beaker and heat to 50-60°C.
2. Add 1.5 g wheat germ and mix until dissolved.
3. Add 5 ml detergent. Maintain 50-60°C temperature and stir for 5 minutes.
4. Add 3 g meat tenderizer.
5. Add baking soda solution to bring the pH to approximately 8.0.
6. Maintain the 50-60°C temperature and stir for 10 minutes.
7. Remove from heat.
8. Add 2 ml of the solution to a test tube and cool to room temperature.
9. Pour 2ml ice cold ethanol carefully down the side of the tube to form a
layer.
11. The DNA will float in the alcohol. Swirl a glass stirring rod at the
interface of the two layers to see the small threads of DNA.
Lima Bean Bacteria DNA Extraction
Protocol
1. Add 14 ml of the bacterial suspension to a centrifuge tube and spin in a

balanced centrifuge for 5 minutes.
2. Pour off the liquid (supernatant) and discard. You want to keep the pellet
as this has your cells.
3. Add 5 ml of prep buffer and re-suspend your cells with a pipette.
4. Add 1 ml 50% detergent solution.
5. Add 1 ml papaya juice.
6. Add 2 ml salt solution and shake for 2 minutes.
7. Place the tube in the centrifuge and spin for 5 minutes. Make sure the
centrifuge is balanced.
8. Draw off 2 ml of the supernatant (liquid) as this has the DNA and
place it in a clean test tube.
9. Pour 2 ml of ice cold ethanol carefully down the side of the tube.
10. Let the mixture sit undisturbed 2-3 minutes until the bubbling stops.
11. The DNA will float in the alcohol. Swirl a glass rod at the interface of
the two layers. You may see some tiny threads of DNA but are more likely
to see fluffy, white sheared DNA.
Yeast DNA Extraction
Protocol
1. Mix 1 package of dry yeast in a beaker with 40 ml of 50 °C hot tap water to

dissolve the yeast. Keep mixture covered and warm for about 20 minutes.
2. Add 40 ml detergent/salt solution.
12
3. Place mixture in a blender and blend 30 sec-1 minute on high.
4. Pour mixture back into the beaker, add 15 ml of meat tenderizer solution,
and stir to mix.
5. Place 2 ml of mixture into a test tube.
6. Pour 2ml of ice cold ethanol carefully down the side of the tube to form a
layer.
8. You will see a precipitate in the alcohol. Swirl a glass stirring rod at the
interface of the two layers. The precipitate is DNA.
When DNA extractions are performed, you can expect three basic results.
1. No DNA
2. DNA appears fluffy which means it has sheared in the extraction

process
3. DNA appears as thin threads (intact DNA)
Materials and Solution preparation

 fresh onions
 dry yeast
 graduated cylinders (10ml and 100ml)
 15 ml test tube
 blender
 ice cold 95% ethanol
Detergent/salt solution:
 20 ml detergent
 20 g non-iodized salt
 180 ml distilled water
5% meat tenderizer solution:
 5 g meat tenderizer
 95 ml distilled water
Buffer solution:
 57 g granulated sugar
 3 g of MgSO4·7H2O
 1 buffered aspirin (one tablet 100 mg)
 add distilled water for a total volume of 500 ml
Salt solution:
 29.2 g non-iodized salt

 add distilled water for a total volume of 250 ml
Baking soda solution:
 Add baking soda to distilled water until a pH of approximately 8.0 is
reached.
Lima Bean Bacteria Suspension: Place 1-2 handfuls of dry lima beans in a
large jar and fill halfway to the top with distilled water. Cover and sit in a warm
room for 2-3 days. Culturing longer than three days often results in more DNA
but it usually shears. Pour through a strainer and keep the liquid for the
extractions.
14
Molecular biology lab Report
Student name:
Group:
Date:
DNA Extraction Summary Chart
WHEAT
QUESTIONS ONION BACTERIA YEAST
GERM
What are the

cell
characteristics?
What lyses the

cell and
nucleus?
What protects
the DNA?
What
precipitates the
DNA?
Amount of DNA
Description of
DNA
The effect or role of the following reagents or tools:
1. Detergent and salt solutions: the lipid walls broken down
2. Temperature of 60oC is necessary to denature the DNase enzymes that
cause shearing in DNA
3. MgSO4 salts and buffered aspirin to further deactivate the enzymes that
degrade DNA and stabilize the DNA
4. Sodium bicarbonate (baking soda) also is used to buffer the solution.
5. The meat tenderizer or Papaya juice has papain, an enzyme that helps
clean the protein from the DNA.
6. The ethanol is used to precipitate the DNA. In water, DNA is soluble. When
it is in ethanol, it uncoils and precipitates leaving behind the other cell
components that are not soluble in ethanol.
7. Water to solubilize DNA.
8. The blender: causes break down of the cell walls, cell membranes, and
nuclear membranes.
16
Laboratory session No. 3:
Phenol/chloroform Extraction of DNA
Some of the most common DNA extraction methods include organic extraction,
Chelex extraction, and solid phase extraction. These methods consistently yield
isolated DNA, but they differ in both the quality and the quantity of DNA yielded.
When selecting a DNA extraction method, there are multiple factors to consider,
including cost, time, safety, and risk of contamination.
Organic extraction involves the addition of and incubation in multiple different
chemical solutions; Chelex extraction method involves adding the Chelex resin to
the sample, boiling the solution, then vortexing and centrifuging it. The cellular
materials bind to the Chelex beads, while the DNA is available in the
supernatant. Solid phase extraction such as using a spin-column based
extraction method takes advantage of the fact that DNA binds to silica. The
sample containing DNA is added to a column containing a silica gel or silica
beads. The DNA binds to the silica, while the rest of the solution is washed out.
All methods of purification involve five essential steps:
1. Cell breakage.
2. nuclear lysis
3. Removal of protein.
4. Removal of RNA.
5. Concentration of DNA.
Cell Breakage
The best procedure for opening cells and obtaining intact DNA is through
application of chemical (detergents) and/or enzymatic procedures. Detergents
can solubilize lipids in cell membranes resulting in gentle cell lysis. In addition,
detergents have an inhibitory effect on all cellular DNases and can denature
proteins, thereby aiding in the removal of proteins from the solution.
Removal of Protein
1. Removal of proteins from the DNA solution depends on differences in the

physical properties between nucleic acids and proteins in organic solvents.
Nucleic acids are predominantly hydrophilic molecules and are easily
soluble in water. Proteins, on the other hand, contain many hydrophobic
residues making them partially soluble in organic solvents.
The organic solvents commonly used are phenol and chloroform

containing 1 percent isoamyl alcohol. The application of phenol is based
on the following principle: Protein molecules generally contain many
hydrophobic residues, which are concentrated in the center of the
molecule. When an aqueous protein solution is mixed with an equal
volume of phenol, some phenol molecules are dissolved in the aqueous
phase (approximately 20 percent water and 80 percent phenol). Yet the
phenol molecules are extremely hydrophobic. Consequently, they tend to
be more soluble in the hydrophobic cores of the protein than in water. As a
result, phenol molecules diffuse into the core of the protein causing the
protein to swell and eventually to unfold or denature. As a result proteins
are partitioned into the phenol phase leaving the nucleic acids in the
aqueous phase. Nucleic acids do not have hydrophobic groups at all and
are insoluble in the phenol phase.
2. The use of ionic detergents. These detergents, by unfolding the protein,

help to expose hydrophobic regions of the polypeptide chains to phenol
micelles, thereby aiding partitioning of proteins into the phenol phase.
18
3. Enzymatic removal of proteins before phenol extraction. This reduces the
number of extractions needed, thus limiting the loss and shearing of DNA.
Two such enzymes are in use, proteinase K and pronase. Proteinase K
and pronase are usually used in DNA purification procedures at final
concentration of 0.1–0.8mg/ml.
4. Addition of 8-OHQ (8-hydroxyquinoline) to the phenol. This increases the
solubility of phenol in water; it plays the role of an anti-oxidant, protecting
phenol against oxidation.
Removal of RNA
The removal of RNA from DNA preparations is usually carried out using two
ribonucleases namely ribonuclease A and ribonuclease T1. Because of the RNA
cleaving specificity of these enzymes, it is recommended that they be used
together for complete RNA removal from DNA samples.
Concentrating the DNA:
1. Ethanol precipitation
2. Phenol–chloroform extraction
3. Mini column purification: that relies on the fact that the nucleic acid may
bind (adsorption) to the solid phase (silica or other) depending on the pH
and the salt content of the buffer. Refinements of the technique include
adding a chelating agent to sequester divalent cations, such as Mg2+ and
Ca2+, which prevents enzymes like DNase from degrading the DNA.
Two alcohols are used for DNA precipitation: ethanol and isopropanol. Alcohol
precipitation is based on the phenomenon of decreasing the solubility of nucleic
acids in water. Polar water molecules surround the DNA molecules in aqueous
solutions. The positively charged dipoles of water interact strongly with the
negative charges on the phosphodiester groups of DNA. This interaction
promotes the solubility of DNA in water. Ethanol is completely miscible with
water, yet, ethanol molecules cannot interact with the polar groups of nucleic
acids as strongly as water, making ethanol a very poor solvent for nucleic acids.
DNA precipitation is customarily carried out with 70 percent ethanol (final
concentration) in the presence of the appropriate concentration of sodium or
ammonium salts. Sodium acetate is more soluble in ethanol than sodium chloride
and, therefore, is less likely to precipitate with the DNA sample.
Materials:
Phenol: chloroform (1:1)
Chloroform
1. Add an equal volume of buffer-saturated phenol: chloroform (1:1) to the

human Blood.
2. Mix well. High molecular weight DNA should be gently rocked.
3. Spin in a microfuge for 3 min.
4. Carefully remove the aqueous layer to a new tube, being careful to avoid
the interface. (Steps 1-4 can be repeated until an interface is no longer
visible).
5. To remove traces of phenol, add an equal volume of chloroform to the
aqueous layer.
6. Spin in a microfuge for 3 min.
7. Remove aqueous layer to new tube.
8. Ethanol precipitate the DNA
Ethanol Precipitation of DNA

3 M sodium acetate (pH 5.2)
100% ethanol
70% ethanol
20
1. In a 1.5 ml Eppendorf tube, take 0.5 ml of the aqueous phase from the
step No. 7. Adjust the salt concentration by adding 1/10 volume of
sodium acetate (50 ul). Add 0.5 ml of 100% ethanol. Mix well.
2. Place on ice or at -20 °C for ~ 15 minutes.
3. Spin a 9000 rpm in a microfuge for 10min.Carefully decants the

supernatant. Add 0.5 ml of ice cold 70% ethanol. Mix. Spin briefly.
Carefully decant supernatant. Air dries the pellet.
4. Resuspend pellet in the 50 ul of sterile distilled water.
5. Store (well labeled eppendorf tubes) in deep freeze to the next week
Determination of the Purity and Quantity of DNA
Introduction
The DNA isolated from living cells are usually contaminated with protein, RNA,
and salts used during the isolation process. The purity of DNA may be estimated
by utilizing the property of the heterocyclic rings of the nucleotides of absorbing
light strongly in the UV range in an Ultraviolet (UV) spectrophotometer. The DNA
has maximum and minimum absorbance at 260 and 234 nm, respectively. The
relationship between DNA absorbance at 260nm (A260) and DNA concentration
(N) is described by the following equation:
DNA concentration (N) = A260 / ε260……………………….(1)
Where ε260 is the DNA extinction coefficient. This coefficient for double stranded
DNA (dsDNA) is 0.02 µg /cm when measured at neutral or slightly basic pH.
Thus, an absorbance of 1.0 at 260 nm gives a DNA concentration of 50 µg /ml
(1/0.02 = 50 µg /ml). As a result the concentration of DNA solutions having an
absorbance of 1.0 is not always a 50 µg/ml.
N = 1 / 0.02 = 50 µg /ml ……………………………………..(2)
The absorption coefficient of single-stranded DNA is 0.027 µg/cm giving an

ssDNA concentration of 37 µg/ml for an absorbance of 1.0 (1/0.027= 37 µg/ml).
Reliable measurements of DNA concentration can be made for solutions of 0.5–

100 µg/ml using a standard UV spectrophotometer. Before measurement,
22
samples with an absorbance equal to or greater than 2.0 should be diluted. The
measurement of DNA concentration at a lower range (A lower than 0.2) can be
strongly affected by light scattering on dust particles present in the preparation.
Measuring the absorbance at 320 nm will assess the degree of such
contamination. At this wave-length, DNA does not absorb and any absorbance
at 320 nm is due to light scattering.
An absorption of 1.0 is equivalent to approximately:
50 μg/mL double-stranded DNA (dsDNA)

33 μg/mL single-stranded DNA (ssDNA)
40 μg/mL single-stranded RNA
30 μg/mL for ssDNA oligonucleotides.
Absorbance measurements at wavelengths other than 260 nm are used for

determination of the degree of protein contamination of the DNA sample.
Proteins absorb maximally at 280 nm due to the presence of tyrosine,
phenylalanine, and tryptophan and absorption at this wavelength is used for the
detection of protein in DNA samples. This is done by determination of the A260 :
A280 ratio. This ratio for pure double-stranded DNA is not 1.8–1.9, as previously
thought, but is 2.0. If the absorbance ratio of 260 nm: 280nm is lower than 2.0,
the DNA concentration can be calculated using following formula:
N (µg /ml= 70 A260 - 40 A280 ………………..(3)
Where A260 and A280 are the absorbances of a DNA sample at 260 and 280 nm,
respectively.
A better indicator of protein contamination in DNA samples is the ratio of A260 /
A234 . DNA has an absorbance minimum at 234 nm and protein absorbance is
high due to the absorption maximum for peptide bonds at 205 nm . Since the
ratio of the DNA extinction coefficient at 234 nm (A234) to the protein extinction
coefficient at the 234 same wavelengths is 1.5–1.8, the A260 / A234 ratio is a very
sensitive indicator of protein contamination. For pure nucleic acids, this ratio is
between 1.8 and 2.0.
Materials
Sample DNA
TE buffer: - Tris-HCI, 10 mM EDTA, 1 mM; pH =7.
Spectrophotometer and quartz cuvette
Procedure
1. To find out the purity of DNA, make the appropriate dilution (1:100) with
TE buffer or DW (e.g., dilute 10 µl DNA+ 990 µl with TE buffer or DW) .
2. Measure the absorbance at 260 nm and 280 nm.
Notes: Use only Quartz cuvettes, do not use glass or plastic cuvettes, as
lights in the UV range do not pass through these.
3. Calculate the DNA concentration as follows:
A. DNA Concentration (µg/ml) = A260 × 50 × dilution factor
B. N (µg /ml= 70 x A260 - 40 x A280
C. DNA purity = 260 nm: 280nm
= A260 / A234 ratio
24
Determination of DNA Melting Point and GC content
DNA denaturation and renaturation:
Double helical DNA is the native (or normal) secondary structure. The
conversion of double helical DNA into single strands is called denaturation or
DNA melting. The conversion of single strands back to the double-stranded
structure is called renaturation or annealing. Annealing is the spontaneous re
association of the separated complementary two single stranded DNA molecules
to form double helical DNA molecule when the temperature is reduced below the
melting temperature.
Melting: Is separation of the two DNA strands in the DNA double helix when the
H bonds between paired bases are broken to generate single stranded DNA.
Melting Temperature (Tm): Is the temperature at which the change in A260 is

half maximal or is 50% of the DNA is double-stranded.
Conditions favoring denaturation or DNA melting include:
 High temperature
 Low salt concentrations. The DNA becomes more negatively charged
resulting in greater strand repulsion. DNA is a polyanionic molecule. The
salt "shields" the negative charges on each phosphate. When the charges
are NOT shielded, the electrostatic repulsion causes strand separation).
The standard salt condition is 0.12 M sodium phosphate buffer (= 0.18 M
sodium ion).
 High pH: DNA can be denatured by heat or alkali (for example, NaOH;
however, RNA is hydrolyzed by alkali).
 Enzymes: helicases
Monitoring DNA denaturation and renaturation
Principle:
When a dilute aqueous DNA solution is heated slowly, the two strands of the
double helix gradually separate, leading to the formation of a single stranded
DNA (denaturation). It results in an increase in absorbance at 260 nm.
Temperature for midpoint of denaturation gives Tm by increasing the
temperature slowly and measuring absorbance at 260 nm as melting profile can
be generated. The DNA of each species has a specific denaturation curve which
is dependent on the % GC content and length. In double stranded DNA, G and C
base pairing is more stable and requires more heat energy to break the three
hydrogen bonds to separate the strands.
26
 Conjugated bonds of bases cause DNA to absorb ultraviolet (UV) light at
260 nm (A260).
 single-stranded DNA is hyperchromic and has higher absorbance at
260nm than double-stranded DNA
 Free nucleotides are even more hyperchromic.
The Tm of DNA sample is affected by a number of factors:
1. Concentration of DNA
2. Concentration of ions in the solution, most notably Mg+ and K+
3. Length of DNA
4. DNA sequence
The most complex factor is the sequence of the DNA. Double-stranded DNA with
a high GC-content has a higher Tm than DNA with a lower GC-content or those
with higher AT ratio for a number of reasons:
 The nucleotide pair ‘A-T’ has a weaker bond than the nucleotide pair ‘G-
C’(The GC pair is bound by three hydrogen bonds, while AT pairs are
bound by two hydrogen bonds).
 Nucleotides on the same strand can interact with each other, forming so-
called secondary structures such as internal loops; these structures
compete with the formation of the double helix and can thus increase the
Tm
 Neighboring nucleotide GC pairs can interact with each other. It is
energetically favorable for nucleotide pairs to be neighbored with other
nucleotide pairs.
 This so-called stacking effect that decreases the Tm. The hydrogen bonds
do not stabilize the DNA significantly, and stabilization is due mainly to
stacking interactions of the DNA base pairs.
The separation of the two DNA strands is also dependent upon the salt
concentration of the DNA solution. To standardize this, all Tm measurements are
made in sodium citrate-sodium chloride buffer (SSC). DNA melts between 85°
and 100° C in this buffer (as opposed to 25° C in distilled water).
Protocol:
Materials
 DNA
 SSC (20x SSC: 0.3M Na(3) citrate , 3M NaCl):
 UV spectrophotometer (preferably with temperature control)
Procedure
1. Place the dissolved DNA in an appropriate quartz cuvette along with a

second cuvette containing SSC as a blank.
2. Place the cuvettes into a water bath at 25 ° C and allow to temperature
equilibrate. Remove the blank, wipe the outside dry and rapidly blank the
instrument at 260 nm. Transfer the sample to the spectrophotometer (be
sure to dry and work rapidly) and read the absorbance.
3. Raise the temperature of the bath to 50° C and repeat step 2
4. Raise the temperature sequentially to 60° C, 65° C, 70° C, 75° C and 80°
C and repeat the absorbance measurements.
5. Slowly raise the temperature above 80° and make absorbance
measurements every 2° until the absorbance begins to increase. At that
point, increase the temperature, but continue to take readings at 1° C
intervals.
6. Correct all of the absorbance readings for solvent expansion relative to 25°
C.
28
7. Plot the value of A at each temperature /A at 25 versus temperature and
calculate the midpoint of any increased absorbance. This midpoint is the
melting point (Tm) for your DNA sample
Determination of GC content of DNA
GC ratios within a genome are found to be markedly variable. Thus

determination of ratio of these specific regions contributes in mapping gene-rich
regions of the genome.
Principle:
GC-content (or guanine-cytosine content) is the percentage of nitrogenous
bases on a DNA molecule that are either guanine or cytosine (from a possibility
of four different ones, also including adenine and thymine).
Three of the most popular methods for obtaining percent guanine plus cytosine
are:
1. The Buoyant Density method
2. The Differential Scanning Calorimetry.
3. The Thermal Denaturation method
In Thermal Denaturation, the melting temperature of the DNA double helix is

measured using spectrophotometry. The absorbance of DNA at a wavelength of
260 nm increases fairly sharply if the double-stranded DNA separates into two
single strands when it is sufficiently heated. This is referred to as
"hyperchromicity”. The higher the %G+C of DNA, the higher is the melting
temperature, and vice versa. This is because the A base pairs with T by two
hydrogen bonds whereas G pairs by three hydrogen bonds with Moreover, the
adjacent GC interacts strongly with each other than adjacent AT base pair.
The temperature is plotted against the absorbance. After this the midpoint is
found, it is called the melting temperature (Tm).
Chargaff's rules: (Base Pair Rule)
It states that DNA from any cell of all organisms should have a 1:1 ratio
of pyrimidine to purine bases and, more specifically, that the amount
of guanine is equal to cytosine and the amount of adenine is equal to thymine.
The base composition of any given DNA is therefore uniquely determined by the
CG-content (or AT-content). It does not apply to organelle DNA
30
(mitochondria and plastids) smaller than ~20-30 kbp, nor does it apply to single
stranded DNA (viral) genomes or any type of RNA genome.
Organism %GC %AT

φX174 44.8 55.2
Rat 42.9 57.0
Human 40.7 59.3
Wheat 45.5 54.4
Yeast 35.8 64.4
E. coli 51.7 48.3
Mathematically, Chargaff's rules can be expressed as:

A+T+G+C=1
A = T, and G = C, where A, T, G, and C are given in mole fractions.
The GC content is usually expressed as a percentage value, but sometimes as a
ratio (called G+C ratio or GC-ratio). The GC-content percentage is calculated as
G+C / G+C+A+T X 100
Whereas, the AT/GC ratio is calculated as:
A+T/G+C
Find out the GC content of your sample using the following formula:
(G + C)% = (Tm - 69.3) x 2.44
Melting temperature also can be calculated from the following equation:
Tm = 81.5 + 16.6(log10 ([Na+])) + .41*(%GC) – 600/length of DNA
[Na conc. =200mM].

Agarose Gel Electrophoresis of DNA
Uses of Gel Electrophoresis
Human DNA can be analyzed by gel electrophoresis to provide evidence in

criminal cases, to diagnose genetic diseases, and to solve paternity cases.
Samples can be obtained from any DNA-containing tissue or body fluid, including
cheek cells, blood, skin, hair, and semen. In many
analyses, polymerase chain reaction (PCR) is used to
amplify specific regions of DNA that are known to vary
among individuals. A person’s “DNA fingerprint” or
“DNA profile” is constructed by using gel electrophoresis
to separate the DNA fragments from several of these
highly variable regions.
Theory of electrophoresis
When a molecule is placed in an electric field it will

migrate to the appropriate electrode with a velocity or free electrophoretic
mobility (M), which is described by the equation:
𝑬 𝒒
𝐌= ∗ ……….(1)
𝒅 𝟔𝛑 𝒓 𝜼
Where:
E: is the potential difference between electrodes

q: is the net charge of the molecule
d: is the distance between electrodes (cm)
𝜂 is the viscosity of the solution
32
r is Stock’s radius of the molecule
E/d is the field strength
Since under physiological conditions phosphate groups in the phosphor sugar
backbone of DNA (RNA) are ionized, these polyanions will migrate to the positive
electrode (anode) when placed in an electric field. Due to the repetitive nature of
the phosphor sugar backbone, double-stranded DNA molecules have a net
charge to mass ratio that is approximately the same. Consequently, DNA
molecules have approximately the same free electrophoretic mobility (M)
irrespective of their size. It is apparent from equation (1) that the effects of friction
on the mobility of the molecules can be accentuated by changing the viscosity (𝜂)
of the electrophoretic medium. Varying the pore size using various agarose
concentrations or different cross-linking ratios of polyacrylamide gel alters the
viscosity of these materials. The mobility of DNA molecules is profoundly
influenced by the size of the gel matrix pores. The electrophoretic migration rate
of DNA through the gel depends on the following parameters:
1. the size of the DNA molecules

2. the concentration of agarose or polyacrylamide gel
3. the voltage applied
4. the conformation of the DNA
5. The type of buffer used for electrophoresis.
DNA molecules travel through gel at a rate inversely proportional to the logarithm
of their molecular weight or number of base pairs. Therefore, a plot of mobility
against the log of the size should give a straight line for all DNA sizes. However,
this is true for a narrow size range. A better linear relationship between mobility
and DNA size is obtained in plots of DNA base pair number (DNA size) versus
1/mobility. The useful linear range of mobility depends on the gel concentration
used and voltage applied. A DNA fragment of a given size migrates at different
rates in gels containing different concentrations of agarose.
The field strength applied to most gels should be between 0.5 and 10V/cm. In
general, higher resolution is achieved at a low voltage gradient, particularly if
higher molecular weight DNA is used. The amount of DNA in a sample will also
affect its apparent mobility
Agarose is a polysaccharide consisting of basic agarobiose repeat units of 1, 3-

linked β-D-galactopyranose and 1, 4-linked 3, 6-anhydro-α-l-galactopyranose
Units form long chains of approximately 400 repeats, reaching a molecular
weight of approximately 120,000 Dalton. Long polymer chains contain small
amounts of charged residues consisting largely of pyruvate and sulfate that are
responsible for the phenomenon of electroendosmosis. During electrophoresis
only hydrated positive ions, which are normally associated with the fixed anionic
groups of agarose (pyruvate or sulfate residues), can move towards the cathode.
Water is therefore pulled with these positive ions towards the negative electrode
and negative molecules, such as DNA migrating towards the positive electrode,
are slowed down. Thus, for maximum separation of DNA molecules by agarose
gel electrophoresis, agarose with the lowest possible electro endosmosis should
be used.
Agarose % of resolution of linear DNA fragments (kb)

0.5 30 to 1
0.7 12 to 0.8
1.0 10 to 0.5
1.2 7 to 0.4
1.5 3 to 0.2
0.5 30 to 1
0.7 12 to 0.8
34
Electrophoresis buffers
Several different buffers are used for agarose gel electrophoresis. These are
TAE (Tris-acetate EDTA) buffer, TBE (Tris-borate EDTA) buffer, and TPA (Tris-
phosphate EDTA) buffer. The ratio of voltage applied to current (mA) is
approximately 1.0 for a wide variety of gel sizes .The of DNA is mixed with
sample loading dye which contains tracking dye, bromophenol blue, which co-
migrates with the smallest DNA molecules at each agarose concentration. In
general, DNA bands are sharper when TBE buffer is used, but the time of
electrophoresis is considerably longer.
Agarose gel electrophoresis is commonly carried out using submerged

horizontal slab gels. The best separation between DNA bands is achieved in
gels that are approximately 4 mm thick. To obtain maximum resolution of many
bands electrophoresis should be continued until the tracking dye (for example
bromophenol blue) has moved 70–80 percent of the length of the gel.
Sample concentration: The amount of DNA loaded into one well should not
exceed 10 mg.
Sample loading dye solutions
DNA samples are prepared for electrophoresis by the addition of loading dye
solution. The loading dye solution plays an essential role in obtaining sharp DNA
bands. This solution serves three vital functions:
1. EDTA: it is used to terminate enzymatic reactions before electrophoresis

(stop solution)
2. Glycerol ,sucrose, Urea or Ficoll 400: it provides density for loading the
sample into the well
3. Xylene cyanol and bromophenol blue dyes: provide a way of monitoring
the progress of electrophoresis.
Gel staining
In order to visualize DNA, agarose gels are usually stained with ethidium
bromide. This is the most rapid, sensitive, and reproducible method currently
available for staining single- and double-stranded DNA. Ethidium bromide binds
to double-stranded nucleic acid by intercalation between stacked base pairs.
Ultraviolet (UV) irradiation of ethidium bromide at 302 and 366 nm is absorbed
and re-emitted as fluorescence at 590nm. The best staining results are obtained
by incorporating ethidium bromide into the gel at a concentration of 0.5 μg/ml.
Photographing gels
Photographs of the gels permit analysis of the data and visualization of DNA
bands .Polaroid cameras, equipped with appropriate filters or computer imaging
system equipped with a charge-couple device digital camera. A DNA band
containing 0.1–4 ng d into a well of 1 cm can be visualized.
Protocol
1. Seal the opened ends of the gel-casting tray with tape. Check that the
teeth of the comb are approximately 0.5 mm above the gel bottom.
2. Place 150 ml of the buffer into a 500 ml flask and add the appropriate
amount of agarose. Weigh 1.5 g of agarose for a 1 percent agarose gel.
Melt the agarose by heating the solution in a microwave oven at full power
for approximately 3 minutes. Carefully swirl the agarose solution to ensure
that the agarose is dissolved that is no agarose particles are visible. Cool
the agarose solution to approximately 60°C and add 2 ul of ethidium
bromide stock solution. Slowly pour the agarose into the gel-casting tray.
Remove any air bubbles by trapping them in a 10 ml pipette.
36
3. Position the comb approximately 1.5 cm from the edge of the gel. Let the
agarose to solidify for approximately 30–60 minutes. Remove the comb
with a gentle back and forth motion, taking care not to tear the gel.
4. Remove the tape from the ends of the gel-casting tray and place the tray
on the central supporting platform of the gel box. Add electrophoresis
buffer to the buffer chamber until it reaches a level of 0.5–1 cm above the
surface of the gel.
5. Load the samples into the wells slowly, allowing it to sink to the bottom of
the well. Take care not to spill the sample into a neighboring well. During
sample loading, it is very important to avoid placing the end of the tip into
the sample well or touching the edge of the well with the tip. This can
damage the well, resulting in uneven or smeared bands.
6. First load 8 μl of the 1kb ladder standard DNA. Next load the entire sample
(10μl)
7. Place the lid on the gel box and connect the electrodes. DNA will travel
towards the positive (red) electrode positioned away from the edge of the
laboratory bench. Turn on the power supply. Adjust the voltage to
approximately 1 V/ cm. For example, if the distance between electrodes
(not the gel length) is 40 cm the voltage should be set to 40 V.
8. Continue electrophoresis until the tracking dye moves at least two- thirds
of the gel length.
9. Turn the power supply off and disconnect the positive (red) lead from the
power supply. Remove the gel from the electrophoresis chamber.
10. Wrap the gel-casting tray with saran wrap and store in a 4°C refrigerator,
or can be photographed immediately.
Principle of the PCR

The purpose of a PCR (Polymerase Chain Reaction) is to make a huge number
of copies of a gene.
PCR is a rapid, inexpensive and simple way of copying specific DNA fragments
from minute quantities of source DNA material, even when that source DNA is of
relatively poor quality. It does not necessarily require the use of radioisotopes or
toxic chemicals.
PCR involves preparation of the sample, the master mix and the primers,
followed by detection and analysis of the reaction products.
Applications of PCR:
1). Medical applications

A. Genetic testing, for the presence of genetic disease mutations.
B. Tissue typing, vital to organ transplantation.
C. Many forms of cancer involve alterations to oncogenes.
2). Infectious disease applications
A. The Human Immunodeficiency Virus (or HIV), Infections can be

detected earlier,
B. Some disease organisms, such as that for Tuberculosis

C. The spread of a disease organism through populations
of domestic or wild animals that were responsible for earlier epidemics
3). Forensic applications
A. Genetic fingerprinting can uniquely discriminate any one person from the
entire population of the world.
38
B. Less discriminating forms of DNA fingerprinting can help in Parental
testing, where an individual is matched with their close relatives.
4). Research applications
A. Hybridization probes for Southern or northern blot hybridization.

B. The task of DNA sequencing can also be assisted by PCR.
C. PCR has numerous applications to the more traditional process of DNA
cloning.
D. Sequence-tagged sites are process where PCR is used as an indicator
that a particular segment of a genome is present in a particular clone.
The Human Genome Project found this application vital to mapping the
cosmid clones they were sequencing, and to coordinating the results from
different laboratories.
E. An exciting application of PCR is the phylogenic analysis of DNA
from ancient sources,
F. A common application of PCR is the study of patterns of gene
expression. This application can also use quantitative PCR to quantitate
the actual levels of expression
G. Genetic mapping by studying chromosomal crossovers after meiosis.
Typical components of a PCR include:
 DNA: the template used to synthesize new DNA strands.

 DNA polymerase: an enzyme that synthesizes new DNA strands.
 Two PCR primers: short DNA molecules (oligonucleotides) that
define the DNA sequence to be amplified.
 Deoxynucleotide triphosphates (dNTPs): the building blocks for the
newly synthesized DNA strands.
 Reaction buffer: a chemical solution that provides the optimal
environmental conditions.
 Magnesium: a necessary cofactor for DNA polymerase activity.
The cycling reactions:

There are three major steps in a PCR, which are repeated for 30 or 40 cycles.
This is done on an automated cycler, which can heat and cool the tubes with the
reaction mixture in a very short time.
1. Denaturation at 94°C: During the denaturation, the double strand melts

open to single stranded DNA, all enzymatic reactions stop (for example:
the extension from a previous cycle).
2. Annealing at 54°C: The primers are jiggling around, caused by the
Brownian motion. Ionic bonds are constantly formed and broken between
the single stranded primer and the single stranded template. The more
stable bonds last a little bit longer (primers that fit exactly) and on that little
piece of double stranded DNA (template and primer), the polymerase can
attach and starts copying the template. Once there are a few bases built in,
the ionic bond is so strong between the template and the primer, that it
does not break anymore.
3. Extension at 72°C: This is the ideal working temperature for the
polymerase. The bases (complementary to the template) are coupled to
the primer on the 3' side (the polymerase adds dNTP's from 5' to 3',
reading the template from 3' to 5' side, bases are added complementary to
the template). Because both strands are copied during PCR, there is
an exponential increase of the number of copies of the gene. Suppose
there is only one copy of the wanted gene before the cycling starts, after
one cycle, there will be 2 copies, after two cycles, there will be 4 copies,
and three cycles will result in 8 copies and so on.
40
Figure 1: The different steps in PCR.
Figure 2: The exponential amplification of the gene in PCR.

Some types of PCR
Multiplex PCR: Multiplex PCR employs different primer pairs in the same
reaction for simultaneous amplification of multiple targets.
Reverse transcription polymerase chain reaction (RT-PCR), a variant

of polymerase chain reaction (PCR), is a technique commonly used in molecular
biology to detect RNA expression. RT-PCR is used to qualitatively detect gene
expression through creation of complementary DNA (cDNA) transcripts from
RNA by reverse transcriptase enzyme.
Real-time polymerase chain reaction (qPCR) is used to quantitatively measure

the amplification of DNA using fluorescent dyes. QPCR is also referred to as
quantitative PCR, quantitative real-time PCR and real-time quantitative PCR. It is
also utilized for quantification of RNA, in both relative and absolute terms. The
combined technique, described as quantitative RT-PCR or real-time RT-PCR
Methylation-specific PCR (MSP): MSP enables the methylation status of target

DNA to be determined after sodium bisulfite treatment. The method requires two
sets of primers to be designed: one set that anneals to unchanged cytosines (i.e.,
methylated in the genomic DNA) and one set that anneals to uracil resulting from
bisulfite treatment of cytosines not methlyated in the genomic DNA.
Factors Affecting the PCR:
1. Denaturing Temperature and time
One may make nucleic acid (NA) single-stranded for the purpose of annealing by
heating it to a point above the "melting temperature" of the double- or partially-
double-stranded form, Additionally, if the NA is heated in buffers of ionic strength
42
lower than 150mM NaCl, the melting temperature is generally less than 100oC -
which is why PCR works with denaturing temperatures of 91-97oC.
Taq polymerase is given as having a half-life of 30 min at 95oC, which is partly

why one should not do more than about 30 amplification cycles. Time at
temperature" is the main reason for denaturation / loss of activity of Taq. Innis
and Gelfand (1990) recommend 96oC for 15 sec.
2. Annealing Temperature and Primer Design
3. Primer Length
4. Elongation Temperature and Time
5. Cycle Number: The number of amplification cycles necessary to produce a
band visible on a gel depends largely on the starting concentration of the target
DNA: Innis and Gelfand (1990) recommend from 40 - 45 cycles to amplify 50
target molecules, and 25 - 30 to amplify 3x105 molecules to the same
concentration. This non-proportionality is due to a so-called plateau effect,
which is the attenuation in the exponential rate of product accumulation in late
stages of a PCR, when product reaches 0.3 - 1.0 nM. This may be caused by
degradation of reactants (dNTPs, enzyme); reactant depletion (primers, dNTPs -
former a problem with short products, latter for long products); end-product
inhibition (pyrophosphate formation); competition for reactants by non-specific
products; competition for primer binding by re-annealing of concentrated (10nM)
product .
If desired product is not made in 30 cycles, take a small sample (1ul) of the
amplified mix and re-amplify 20-30x in a new reaction mix rather than extending
the run to more cycles: in some cases where template concentration is limiting,
this can give good product where extension of cycling to 40x or more does not.
PCR controls
1. No-template control: The NTC reaction contains all PCR components

except the template. Detection of a positive signal in an NTC reaction
indicates the presence of contaminating nucleic acids.
2. Positive control: A positive control can be an absolute standard, which is
a nucleic acid from an established cell line, a plasmid containing cloned
sequences, or in vitro transcribed RNA, .A positive control can also be a
known positive sample, which is usually a substitute for an absolute
standard and used only to test for the presence or absence of a target.
3. No RT control: where real-time RT-PCR is carried out without reverse
transcriptase, should be included when performing gene expression
analysis.
Contaminating DNA in RNA samples can be removed by DNase treatment
before starting RT-PCR.
4. Internal controls: An internal, positive control can be used to test for the
presence of PCR inhibitors. A duplex reaction is carried out, where the
target sequence is amplified with one primer–probe set, and a control
sequence (i.e., the internal, positive control) is amplified with a different
primer–probe set. The internal, positive control should be at a high enough
copy number for accurate detection. If the internal, positive control is
detected, but the target sequence is not, then this indicates that the
amplification reaction was successful and that the target sequence is
absent (or at too low a copy number to be detected).
44
Detection and analysis of the reaction product
Take 1/10th - 1/3rd of the reaction mix this with some gel loading buffer (1:1 - 1:5
mix: loading buffer): this is TBE containing 10 - 20% glycerol or sucrose and a
dash of bromophenol blue (BPB) tracking dye. Load 5 - 30ul of sample into wells
of 0.8 - 3.0% submarine agarose gel made up in TBE, preferably containing
50ng/ml ethidium bromide. Run at 80 -120 volts (not too slow or small
products diffuse; not too fast or bands smear) until BPB reaches end of gel (large
products) or 2/3 down gel (small products). Use DNA markers going from 2kb
down to 100 bp or less (recommend BM PCR markers). View on UV light box at
254 - 300 nm, photo 1 - 5 sec.
Figure 5: Verification of the

PCR product on gel.
Lane 1: PCR fragment is
approximately 1850 bases long.
Lane 2 and 4: the fragments
are approximately
800 bases long. Lane 3: no
product is formed, so the PCR
failed.
Lane 5: multiple bands are
formed because one of the
primers fits on different places.
45
Annealing Temperature and Primer Design
Primers:
A primer is a short segment of nucleotides which is complementary to a section
of the DNA which is to be amplified in the PCR reaction. Primers are annealed to
the denatured DNA template to provide an initiation site for the elongation of the
new DNA molecule. Primers can either be specific to a particular DNA
nucleotide sequence or they can be "universal." Universal primers are
complementary to nucleotide sequences which are very common in a particular
set of DNA molecules. Thus, they are able to bind to a wide variety of DNA
templates.
Bacterial ribosomal DNA genes contain nucleotide sequences that are common
to all bacteria. Thus, bacterial universal primers can be made by creating primers
which are complementary to this sequences. The universal primer sequence is
as follows:
5' GTG GAT CAC CTG AGG TCA GGA GTT TC 3' (26 mer)
PCR primer design

Optimal primer sequences and appropriate primer concentrations are essential
for maximal specificity and efficiency in PCR. The table, Guidelines for the design
and use of primers provides an overview of primer design and use for standard
and multiplex PCR, as well as one-step RT-PCR. Molar conversions can be
found in the table Molar conversions for PCR primers.
46
The following points should be considered when designing PCR primers and are
common to all types of PCR:
 primers should be 17-28 bases in length;
 base composition should be 50-60% (G+C);
 primers should end (3') in a G or C, or CG or GC: this prevents "breathing"
of ends and increases efficiency of priming;
 Tms between 55-80oC are preferred;
 Tm calculation:
2°C x (A+T) + 4°C x (G+C)
 Avoid complementarity in the 2–3 bases at the 3' end of the primer pairs
 Avoid runs of 3 or more Cs or Gs at the 3' end of the primer ,this may
promote mispriming at G or C-rich sequences (because of stability of
annealing), and should be avoided;
 Avoid complementarity within primers and between the primer pair
 Avoid a T as ultimate base at the 3' end
 Ensure primer sequence is unique for the template sequence
 Use a concentration of 0.1–1.0 µM of each primer. For many applications,
a primer concentration of 0.2 µM will be sufficient
 3'-ends of primers should not be complementary (ie. base pair), as
otherwise primer dimers will be synthesized preferentially to any other
product;
Lyophilized primers should be dissolved in a small volume of distilled water or TE

to make a concentrated stock solution. Prepare small aliquots of working
solutions containing 10 pmol/µl to avoid repeated thawing and freezing. Store all
primer solutions at –20°C. Primer quality can be checked on a denaturing
polyacrylamide gel; a single band should be seen.
47
Annealing Temperature and Primer Design
Melting temperature of NA duplex increases both with its length, and with
increasing (G+C) content: a simple formula for calculation of the Tm of the primer
is
Tm = 4(G + C) + 2(A + T)oC
Thus, the annealing temperature chosen for a PCR depends directly on length
and composition of the primer(s).
Annealing temperature (Ta) = Tm- 5oC
One consequence of having too low a Ta is that one or both primers will anneal
to sequences other than the true target
A consequence of too high a Ta is that too little product will be made.
Annealing does not take long: most primers will anneal efficiently in 30 seconds
or less
Primer Dimer
48
Hairpin loop self-complementary structure
Labouratory work:
1. Design a FW and Reverse primer set for the following gene?
Homo sapiens albumin gene (part of gene sequence is shown)

1 agtatattag tgctaatttc cctccgtttg tcctagcttt tctcttctgt caaccccaca
61 cgcctttggc acaatgaagt gggtaacctt tatttccctt ctttttctct ttagctcggc
121 ttattccagg ggtgtgtttc gtcgagatgc acacaagagt gaggttgctc atcggtttaa
181 agatttggga gaagaaaatt tcaaagcctt ggtgttgatt gcctttgctc agtatcttca
241 gcagtgtcca tttgaagatc atgtaaaatt agtgaatgaa gtaactgaat ttgcaaaaac
301 atgtgttgct gatgagtcag ctgaaaattg tgacaaatca cttcataccc tttttggaga
361 caaattatgc acagttgcaa ctcttcgtga aacctatggt gaaatggctg actgctgtgc
421 aaaacaagaa cctgagagaa atgaatgctt cttgcaacac aaagatgaca acccaaacct
481 cccccgattg gtgagaccag aggttgatgt gatgtgcact gcttttcatg acaatgaaga
541 gacatttttg aaaaaatact tatatgaaat tgccagaaga catccttact tttatgcccc
49
601 ggaactcctt ttctttgcta aaaggtataa agctgctttt acagaatgtt gccaagctgc
661 tgataaagct gcctgcctgt tgccaaagct cgatgaactt cgggatgaag ggaaggcttc
721 gtctgccaaa cagagactca agtgtgccag tctccaaaaa tttggagaaa gagctttcaa
781 agcatgggca gtagctcgcc tgagccagag atttcccaaa gctgagtttg cagaagtttc
841 caagttagtg acagatctta ccaaagtcca cacggaatgc tgccatggag atctgcttga
901 atgtgctgat gacagggcgg accttgccaa gtatatctgt gaaaatcaag attcgatctc
961 cagtaaactg aaggaatgct gtgaaaaacc tctgttggaa aaatcccact gcattgccga
1021 agtggaaaat gatgagatgc ctgctgactt gccttcatta gctgctgatt ttgttgaaag
1081 taaggatgtt tgcaaaaact atgctgaggc aaaggatgtc ttcctgggca tgtttttgta
2. Determine the Tm, Ta, GC%, and Molecular weight of each primer?
- Note: The average MW. of each nucleotide= 330 Dalton
50
Laboratory session No. 9.
Preparation of PCR Master Mix
It is a PCR reaction buffer called Master Mix that contains all of the components
necessary to make new strands of DNA in the PCR process. The Master Mix
reagents include (table 1):
If want to make 10 reactions, Multiply all components (except DNA template) by

10 total number of the reactions
Solve for the volume of the sterile water needed per reaction by subtracting the
volumes of all other reaction components from 50 ul:
50 µl (final reaction volume) – 10 µl buffer – 5 µl (forward primer) – 5 µl (reverse

primer) – 1 µl (Taq Polymerase) - 4 µl (dTNP) - 5 µl (DNA template) = µl sterile
water
Protocol:
TABLE 1: To make the master mix for one reaction, add:
Volume
For one Final
Master mix components for 10
reaction conc.
reactions
1. 5X PCR buffer 10 µl 100 µl 1X
Each nucleotide mixture,25 mM
2. 4 µl 40 µl 200 µM
(dATP, dCTP, dGTP, dTTP))
3. forward primer 5 µl 50 µl 0.2 µM
51
4. Reverse primer 5µl 50 µl 0.2 µM
1 µl Taq DNA polymerase (
5. 1µl 10 µl 5 units
5U/µl)
6. water 20 µl 200 µl
7. Total volume 45 µl 450 µl
Solutions to be added Volume
Master mix 45µl

DNA template 5 µl
Notes on the Master Mix

The Master Mix buffer is often stored as a 5X stock solution (100 mM Tris-HCL,
pH 8.3, 500 mM KCL, 75 mM MgCl2) which is diluted to 1X for use. Both the
Master Mix buffer and the purified water can be stored at room temperature.
Store the deoxynucleotides, primers and Taq DNA polymerase enzyme at -20oC.
Always Run A No-DNA Control To Check For Contamination
Programming of PCR machine
The thermal cycler (also known as a thermocycler, PCR machine or DNA

amplifier) is a laboratory apparatus most commonly used to amplify segments
of DNA via the polymerase chain reaction (PCR). Thermal cyclers may also be
used in laboratories to facilitate other temperature sensitive reactions,
including but not limited to restriction enzyme digestion or rapid
diagnostics. The device has a thermal block with holes where tubes holding
52
the reaction mixtures can be inserted. The cycler then raises and lowers the
temperature of the block in discrete, pre-programmed steps
Final Conc. Component Purpose

1X Buffer keeps the master mix at
1X Buffer
the proper pH so the PCR reaction will take place
provide both the energy and nucleosides for the
synthesis of DNA. It is important to add equal
200µM dNTP amounts of each nucleotide (dATP, dTTP,
dCTP,GTP) to the master mix to prevent
mismatches of bases.
Short pieces of DNA (20-30 bases) that bind to
the DNA template allowing Taq DNA polymerase
0.2-1.0µM Primers enzyme to initiate incorporation of the
deoxynucleotides.Both specific and universal
primers can be used.
2.5U/100µl A heat stable enzyme that adds the

Taq
deoxynucleotides to the DNA template.
The DNA which will be amplified by the PCR
0.05-1.0µg Template
reaction.
In Programming of PCR machine, Each PCR cycle include:-
Denaturation: Denaturation for 30 seconds at 94-95oC is routinely used to

amplify linear DNA molecules whose GC content is <55% and higher
temperature for template and/or target DNAs whose GC content is >55%.
Annealing of primers to template DNA: Annealing is best to optimized by

performing a series of trial PCRs at temperatures ranging from 2 C to 10 C below
the lower of melting temperatures calculated for the two oligonucleotide primers.
Optimization is achieved by exposing a single PCR to a sequential series of
annealing temperatures in successive cycles of the reaction. The polymerization
rate of Taq polymerase is about 2000 nucleotides /minute at the optimal
temperature (72oC) .
53
Extension of oligonucleotide primers (DNA synthesis): Is carried out at or
near the optimal temperature for DNA synthesis which is catalyzed by the
thermostable polymerase, which in the case of Taq polymerase is 72-78oC.
Number of cycles
The number of cycles required for amplification depends on the number of copies
of template DNA present at the beginning of the reaction and the efficiency of
primer extension and amplification. At least 25 cycles are required to achieve
acceptable levels of amplification of single copy target sequences in mammalian
DNA templates
Stages Temp time cycles
Initial 94 2 minutes 1
denaturation
denaturation 94 15 s
annealing 55 30 s 30 cycles
extension 72 1 minute
Final extension 72 5 minutes
hold 4 0 ( infinity)
54
Spin column method of RNA isolation by acid

guanidinium thiocyanate–phenol–Chloroform
extraction
Several methods are used in molecular biology to isolate RNA from samples, the
most common of these is guanidinium thiocyanate-phenol-chloroform extraction.
The filter paper based lysis and elution method features high throughput
capacity. The RNA extraction procedure is complicated by the ubiquitous
presence of ribonuclease enzymes in cells and tissues, which can rapidly
degrade RNA. RNases, a group of enzymes that degrade RNA molecules, are
abundant in the environment, including on hands and on surfaces and it is
difficult to remove/destroy RNases completely. RNA extraction in liquid nitrogen,
commonly using a mortar and pestle is also useful in preventing ribonuclease
activity.
Methods for RNA extraction need to be tailored to the organism from which the
RNA is being extracted. Plants pose additional challenges due to the presence of
secondary metabolites, polyphenols and polysaccharides. The addition of
polyvinylpyrrolidone (PVP) to the extraction buffer aids the removal of phenolic
compounds and polysaccharides.
For extraction of RNAs from plants, blood and cerebrospinal fluid, there are more
than ten commercially available RNA-isolation kits. There are Magnetic bead-
based RNA extraction modalities, Silica-coated beads for total RNA extraction,
55
and oligo (dT) beads for mRNA extraction. The major categories of RNA
extraction methods are listed in Table 1.
Table 1. Basic steps involved in the RNA extraction using organic solvents
/chaotropic agents.
Cell lysis can be achieved using buffers or
reagents containing chaotropic agents such as
guanidinium isothiocyanate, guanidinium chloride,
Cell lysis and dissolution sodium dodecyl sulphate (SDS), sarcosyl, urea,
phenol or chloroform. TRIzol or RNAlater or Qiazol
can be used to maintain RNA integrity during lysis.
DNase can be used to degrade DNA, while

proteinase K can be added to digest proteins.
Denaturation of DNA and Alternatively, repeated organic extraction using
proteins phenol and chloroform, or dissolving the sample in
buffers containing guanidinium salts, can also be
used to remove proteins.
This can be achieved using any of the chaotropic

Denaturation and
agents mentioned above, such as phenol and
inactivation of RNases
chloroform.
RNA can be separated from other cellular

components by adding chloroform and centrifuging
Removal/separation of
the solution. This separates the solution into two
cellular components
phases: organic and aqueous phases. The
aqueous phase contains RNA.
RNA is often recovered from the aqueous phase

using isopropyl alcohol. RNA can also be
selectively precipitated from DNA through the use
Precipitation
of ammonium acetate. Alternatively, lithium
chloride can be used to selectively precipitate RNA
from DNA as well as proteins
56
Principle for RNA extraction from blood
The miRNeasy Serum/Plasma Kit combines phenol/guanidine-based lysis of

samples and silica-membrane–based purification of total RNA. QIAzol Lysis
Reagent, included in the kit, is a monophasic solution of phenol and guanidine
thiocyanate, designed to facilitate lysis, to denature protein complexes and
RNases, and also to remove most of the residual DNA and proteins from the
lysate by organic extraction.
QIAzol Lysis Reagent is added to serum or plasma samples. After addition of
chloroform, the lysate is separated into aqueous and organic phases by
centrifugation. RNA partitions to the upper, aqueous phase, while DNA partitions
to the interphase and proteins to the lower, organic phase or the interphase.
The upper, aqueous phase is extracted, and ethanol is added to provide
appropriate binding conditions for all RNA molecules from approximately 18
nucleotides (nt) upwards. The sample is then applied to the RNeasy MinElute
spin column, where the total RNA binds to the membrane and phenol and other
contaminants are efficiently washed away. High-quality RNA is then eluted in a
small volume of RNase-free water.
Figure. Basic steps in

RNA extraction using
spin column from
miRNeasy
Serum/Plasma Kit.
57
Procedure
1. Prepare serum or plasma or thaw frozen samples (200µl)

2. Add 5 volumes QIAzol Lysis Reagent (1 ml). Mix by vortexing or pipetting
up and down.
3. Place the tube containing the lysate on the bench top at room temperature
(15–25°C) for 5 min.
4. Add chloroform of an equal volume to the starting sample to the tube
containing the lysate and cap it securely (200 µl l). Vortex or shake
vigorously for 15 sec, thorough mixing is important for subsequent phase
separation. Place the tube containing the lysate on the bench top at room
temperature (15–25°C) for 2–3 min.
5. Centrifuge for 15 min at 12,000 x g at 4°C.
6. After centrifugation, the sample separates into 3 phases: an upper,
colorless, aqueous phase containing RNA; a white interphase; and a
lower, red, organic phase.
7. Transfer the upper aqueous phase to a new collection tube. Avoid transfer
of any interphase material. Add 1.5 volumes of 100% ethanol and mix
thoroughly by pipetting up and down several times. Do not centrifuge.
Continue without delay with step 9.
8. Pipet up to 700 μl of the sample, including any precipitate that may have
formed, into an RNeasy MinElute spin column in a 2 ml collection tube
(supplied). Close the lid gently and centrifuge at> 8000 x g (>10,000 rpm)
for 15 s at room temperature (15–25°C). Discard the flow-through. Reuse
the collection tube in step 9
9. Repeat step 9 using the remainder of the sample. Discard the flowthrough.
Reuse the collection tube in step 10.
58
10. Add 700 μl Buffer RWT to the RNeasy MinElute spin column. Close
the lid gently and centrifuge for 15 s at >8000 x g (>10,000 rpm) to wash
the column. Discard the flow-through. Reuse the collection tube in step 11.
11. Pipet 500 μl Buffer RPE onto the RNeasy MinElute spin column.
Close the lid gently and centrifuge for 15 s at >8000 x g (>10,000 rpm) to
wash the column. Discard the flow-through. Reuse the collection tube in
step 12.
12. Pipet 500 μl of 80% ethanol onto the RNeasy MinElute spin column.
Close the lid gently and centrifuge for 2 min at >8000 x g (>10,000 rpm) to
wash the spin column membrane. Discard the collection tube with the flow-
through.
13. Place the RNeasy MinElute spin column into a new 2 ml collection
tube (supplied). Open the lid of the spin column, and centrifuge at full
speed for 5 min to dry the membrane since residual ethanol may interfere
with downstream reactions. Discard the collection tube with the flow-
through.
14. Place the RNeasy MinElute spin column in a new 1.5 ml collection
tube (supplied). Add 14 μl RNase-free water directly to the center of the
spin column membrane. Close the lid gently, and centrifuge for 1 min at full
speed to elute the RNA. The dead volume of the RNeasy MinElute spin
column is 2 μl: elution with 14 μl RNase-free water results in a 12 μl eluate.
Anticipated Results
The original mini spin column method is expected to yield the whole spectrum of
RNA molecules, including small (4S to 5S) RNAs. The amount of RNA isolated
will depend on the tissue used for isolation. Typically, from 100 μg to 150 μg of
total RNA can be isolated from 100 mg of muscle tissue and up to 800 μg can be
isolated from 100 mg of liver. The yield of total RNA from 1 × 107 cultured cells
59
should range from 5 μg to 80 μg for fibroblasts and lymphocytes and from 100 μg
to 120 μg for epithelial cells.
The A260/A280 ratio of the isolated RNA should be above 1.8. The typical
electrophoretic pattern of RNAs isolated by the single-step method is shown in
the following figure.
60
Analyzing RNA Quality and integrity
The most important and certainly the most often used technique in RNA analysis
is gel electrophoresis. This technique is generally applicable for RNA detection,
quantification, purification by size, and quality assessment. Because RNAs are
negatively charged, they migrate toward the anode in the presence of electric
current. The gel acts as a sieve to selectively impede the migration of the RNA in
proportion to its mass, given that it’s mass is generally proportional to its charge.
Because mass is approximately related to chain length, the length of an RNA is
more generally determined by its migration. In addition, topology (i.e., circularity)
can affect migration, making RNAs appear longer on the gel than they actually
are.
There are two common types of gel: polyacrylamide and agarose. For most
applications, denaturing acrylamide gels are most appropriate. These gels are
extremely versatile and can resolve RNAs from ~600 to ~20 nucleotides (nt). The
disadvantages to acrylamide gels is that they are not suitable for analyzing large
RNAs (~600 nt) , that they are much more difficult to handle than agarose gels
and Acrylamide is a potent neurotoxin. So, agarose gels are preferred. For
experiments requiring separation of large molecules (~600 nt), a method for use
of agarose gels is described in nondenaturing agarose gel electrophoresis of
RNA.
1. RNA analysis on nondenaturing agarose gel electrophoresis.
Agarose concentration
Linear RNA of a given size migrates through agarose of different concentrations
at different rates given by:
Log m = logmo KrT
Where:
61
m is the electrophoretic mobility of the RNA.
mo is the free electrophoretic mobility of the RNA.
Kr is the retardation coefficient.
T is the gel concentration.
The following table reveals the appropriate percentage of the agarose gel matrix
used for the efficient separation of the linear RNA molecules.
% of Efficient range of
agarose separation of linear
(w/v) in gel RNA molecules (bp)
1. 0.3 5 000- 60000
2. 0.6 1 000 -20000
3. 0.7 800- 10000
0.9
4. 500- 7000
5. 1.2 400- 6000
6. 1.5 200- 3000
7. 2.0 100- 2000
The following gel electrophoresis conditions are recommended:

1) use 1X TAE buffer instead of 1X TBE
2) use agarose gel in the concentration of 1.1%-1.5%
3) add ethidium bromide (EtBr) to the gel and electrophoresis buffer to avoid
the additional (potentially RNAse-prone) step of gel staining
4) Always use fresh gel and buffer (filter the buffer with Millipore filter) as well
as clean electrophoresis equipment for RNA analysis by alcohol. Wear
gloves to protect RNA samples from degradation by nucleases and avoid a
hand contact with EtBr.
5) Heat an aliquot of the RNA solution at 70°C for 1-2 min and place it on ice
before loading on a gel.
6) Load a known amount of DNA or RNA ladder alongside your RNA sample
as a standard for determining the RNA concentration.
62
7) For maximum resolution, run agarose gels at no more than 5V / cm
(measured between the electrodes, not the gel length).The use of the
tracking dye will help to optimize the run time which is approximately 20-30
minutes.
8) The first sign of RNA degradation on the nondenaturing gel is a slight
smear starting from the rRNA bands and extending to the area of shorter
fragments. RNA showing this extent of degradation is still good for further
procedures. However, if the downward smearing is so pronounced that the
rRNA bands do not have a discernible lower edge, this RNA should be
discarded.
9) Commonly, genomic DNA contamination does not exceed the amount
seen on the agarose/EtBr gel as a weak band of high molecular weight.
Such contamination does not affect cDNA synthesis. DNase treatment to
degrade genomic DNA is not recommended. In some cases, excess of
genomic DNA can be removed by LiCl precipitation or by phenol:
chloroform extraction.
2. RNA analysis on denaturing agarose gel electrophoresis:

I. Denaturing MOPS buffer/formaldehyde gel electrophoresis
of RNA
Separation of RNA based on fragment length requires conditions that are
different from DNA analysis. These include sample preparation, the use of
sample and gel denaturants, electrophoresis buffers, and visualization.
Principle
Since RNA is a single-stranded nucleic acid and tends to form secondary
structures, a standard agarose gel will not give an accurate size separation
of your total RNA or mRNA sample. Therefore a denaturing agent like
63
formaldehyde must to be added to the agarose gel and the RNA to ensure
the molecules remain single-stranded. Because of the single-stranded
nature of the molecule, RNA tends not to incorporate stains like ethidium
bromide as well as DNA. We recommend including ethidium bromide in
both the loading buffer and the gel to give better staining and visualization
of the RNA. The following is a basic protocol for pouring a denaturing
MOPS buffer/formaldehyde gel and performing electrophoresis of RNA
samples to visually assess the quality of the purified RNA.
Protocol
Reagents and materials
RNA - 10X MOPS Buffer (10X MOPS Buffer (200 mm MOPS, 10 mM EDTA,
50 mM Na Acetate, pH 7.0 NaOH). prepare as follows:
800 mls DDH2O (DEPC)

41.8 g MOPS
Make the pH to 7.0 with NaOH/Acetic Acid
16.6 ml 3M NaAc (DEPC) pH 5.2

20 ml 500 mM EDTA, pH 8.0
Complete the volume up to 1L, Filter into sterile bottle.
37% (v/v) formaldehyde

1X MOPS Buffer
The RNA sample buffer is comprised of:

65% formamide
22% formalin (37% formaldehyde)
13% 10X MOPS
RNA sample Dye: Add 10µl of the following:
50% glycerol
1mM EDTA
0.3% bromophenol blue
0.3% xylene cyanol.
64
Agarose gel electrophoresis
1. Remove an aliquot of 5 µg of the total RNA to a fresh, labeled 1.5 ml

Eppendorf tube on ice. In a 250 ml Erlenmyer flask mix:
0.5 g agarose
36 ml distilled water
-Make the microwave on medium-low for one minute. Bring the melted agarose
to 60°C. . Use DEPC treated water and RNase free reagents.
2. Add 5 ml 10X MOPS Buffer and swirl gently to mix.

3. In a fume hood, add 3 ml 37% formaldehyde solution, swirl to mix and
immediately pour into the gel tray. Close the hood and allow to set for
about 30 minutes. While the gel is hardening, prepare the sample for
loading. Mix the following together on ice:
5 µg RNA ? µl
sterile
distilled ? µl
water
RNA
Sample 10 µl
Buffer
Total volume 20 µl
4. Heat to 65 C for 5 minutes, OR Mix 40µl sample buffer with 10µl

sample, heat to 55°C 15 minutes. cool on ice for 5 minutes
5. Add 2 µl RNA Sample Dye. Vortex for 5 seconds and spin for 5
seconds to pull the sample to the bottom of the tube. Store in your ice
bucket until you are ready to load the gel.
6. Place the gel in the electrophoresis chamber with the wells closest to
the cathode (black [-ve]). The RNA has negatively charged phosphate
65
groups (anions) and will migrate from the cathode to the anode (red
[+ve]).
7. Pour about 350 ml 1X MOPS Buffer into the tank. The gel should be
covered completely.
8. Load your samples. Since the sample is in 22 µl, load it a half at a time
9. The first well should contain a molecular marker mixture. The other
wells should be loaded with the samples.
10. Carefully connect the leads to the power supply. Red connects to
positive, black to negative.
11. Turn on the power supply and set the voltage to 100 volts.
12. After about 10 minutes, check to see that the dye is moving in the
correct direction, towards the anode (red).
13. Allow the gel to run for about one hour or until the bromophenol blue
marker dye is about two-thirds the way down the gel.
14. Turn off the power supply. Wait one minute and remove the leads.
15. Transfer the gel to a tray containing distilled water. Place on a shaker
and shake gently for 10 minutes. Repeat twice, discarding the
formaldehyde-contaminated water to the marked waste container.
16. Add a mixture of 50 ml (1X MOPS Buffer + 5 µl of 10 mg/ml ethidium
bromide) and stain the gel for 10 minutes on the shaker.
17. Destain the gel for 30 minutes in distilled water on the shaker.
18. Carefully transfer the gel to the transilluminator. Short wave
ultraviolet and observe the gel. Destain the gel further if the
background staining is still high.
Photograph the gel with a ruler next to the left had side aligned so that the
zero mark is next to the bottom of the well.
II. Bleach denaturing agarose gel
66
Principle
RNA quality can be quickly analyzed by adding small amounts of commercial
bleach to TAE buffer-based agarose gels prior to electrophoresis. In the
presence of low concentrations of bleach, the secondary structure of RNA is
denatured by destroying hydrogen bonds and potential contaminating RNases
and protein denaturation due to oxidation that occurs after exposure to
hypochlorite (by incorporating common 6% sodium hypochlorite) into a TAE
buffer and agarose mixture prior to melting the agarose.
Protocol
Incubate the solutions at room temperature for 5-10 minutes.
1. 1% TAE agarose ‘bleach gel’ is best run at a concentration of 0.5% to 1%
v/v bleach.
2. The agarose gels were then heated to melt the agarose and cooled. Three
μl of 10 mg/ml ethidium bromide were added to each gel, and the gels
were poured into molds and allowed to solidify.
3. The gels were placed in mini-gel electrophoresis apparatus and
submerged completely with 1× TAE buffer.
4. 10 μl sample containing ~1ug of total RNA mixed with 10× DNA Loading
buffer stock (1.9 mM xylene cyanol, 1.5 mM bromophenol blue, 25%
glycerol in sterile dH20) and is added to each well.
5. The gels were run for ∼35 minutes with constant voltage (100 V) prior to
imaging under UV transillumination.
When assessing the quality of RNA by gel electrophoresis, the presence of
three distinct bands suggests high quality RNA. For eukaryotic RNA, the top
band represents 28S ribosomal RNA (rRNA), which runs at ∼4.8 kb; the
middle band represents 18S rRNA at ∼2.0 kb; and the third band represents
5.8S (154 nt) and 5S (117 nt) RNA. Transfer RNAs (73-93 nt) may or may not
be visible.
67
For mammalian total RNA, two intensive bands should be observed against a
light smear. These bands represent 28S and 18S rRNA. The ratio of intensities
of these bands should be about 1.5-2.5:1. Intact mammalian poly (A)+ RNA
appears as a smear sized from 0.1 to 4-7 (or more) kb with faint 28S and 18S
rRNA bands.
Figure: Protective effect of bleach on RNA quality in agarose gels
68
Estimation of RNA by Orcinol Reaction
Principle:
This is a general reaction for pentoses and depends on the formation of furfural
when the pentose is heated with concentrated hydrochloric acid. Orcinol reacts
with the furfural in the presence of ferric chloride as a catalyst to give a green
color, which can be measured at 665 nm.
Requirements:
1. Standard RNA solution- 200μg/ml in 1 N perchloric acid/buffered saline.
2. Orcinol Reagent- Dissolve 0.1g of ferric chloride in 100 ml of concentrated
HCl and add 3.5 ml of 6% w/v orcinol in alcohol.
3. Buffered Saline- 0.5 mol/liter NaCl; 0.015 mol/liter sodium 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 labeled 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 orcinol reagent to all the test tubes including the test
tubes labeled 'blank' and 'unknown'.
5. Mix the contents of the tubes by vortexing / shaking the tubes and heat on
a boiling water bath for 20min.
6. Then cool the contents and record the absorbance at 665 nm against
blank.
69
7. Then plot the standard curve by taking concentration of RNA along X-axis
and absorbance at 665 nm along Y-axis.
8. Then from this standard curve calculate the concentration of RNA in the
given sample.
Result: The given unknown sample contains ----μg RNA/ml.
Observations and Calculations
Stock DW Working
standard RNA Volume RNA conc.
Volume-ml (ml) (µg/ml)
0 1 00
0.2 0.8 40
0.4 0.6 80
0.6 0.4 120
0.8 0.2 160
1 0 200
70
Nucleotide Composition of RNA
Materials
 RNA sample
 1 N and 0.1 N HCl
 Boiling water bath
 Whatman #1 filter paper (for chromatography)
 Chromatography tank
 20 µl micropipette
 Acetic acid: butanol: water (15:60:25) solvent
 UV light source
 UV spectrophotometer
 10 µg of each nucleotide /50µl of 0.1N HCl
Procedure
1. Place a portion of your RNA sample (approximately 80 mg, 1 ml) into a

heavy walled Pyrex test tube. Add 1.0 ml of 1 N HCl and seal the tube.
2. Heat the tube in a boiling water bath for 1 hour.
3. Cool the tube, open it and place the contents into a centrifuge tube.
Centrifuge the contents at 2,000 RPM in a clinical centrifuge to remove any
insoluble residue. The supernatant contains hydrolyzed RNA (~1.5 ml).
4. Prepare Whatman filter paper No. 1 for standard one- dimensional
chromatography.
5. Using a micropipette, spot 20 µl of your hydrolysate onto the paper, being
careful to keep the spots as small as possible (repeated small drops are
71
better than one large drop). Allow the spots to completely dry before
proceeding.
6. Place the paper chromatogram into your chromatography tank and add the
solvent (acetic acid: butanol: water). Allow the system to function for an
appropriate time (approximately 36 hours for a 20 cm descending strip of
Whatman #1). Remove the paper and dry it in a circulating air oven at 40°
C for about 2 hours.
7. Locate the spots of nucleotides by their fluorescence under a ultra-violet
light source. Expose the paper chromatogram to a UV light source and
outline the spots using a light pencil. The order of migration from the point
of origin is guanine (light blue fluorescence), adenine, cytidylic acid and
finally, uridylic acid.
8. After carefully marking the spots, cut them out with scissors and place the
paper cut outs into separately labeled 15 ml conical centrifuge tubes. Add
2.0 ml of 0.1 N HCl to each tube and allow the tubes to sit for several
hours to elute the nucleotides from the paper.
9. Pack down the paper with a glass rod (centrifuge in a clinical centrifuge if
necessary) and remove an aliquot of the liquid for spectrophotometric
assay.
10. Measure the absorbance of each of the four nucleotides at the indicated
UV wavelength (having first blanked the instrument with 0.1 N HCl).
Base Wavelength Molar Extinction Coefficient

Guanine 250 nm 10.6
Adenine 260 nm 13.0
Cytidylic acid 280 nm 19.95
Uridylic acid 260 nm 9.89
72
Use the molar extinction coefficients to determine the concentration of each base
in the sample. Calculate the percent composition of each base, and the
purine/pyrimidine ratio.
Suppose A260 for adenine is y O.D. units
𝐲(𝐎.𝐃) 𝑽𝟐 𝑽𝟑
∴µmoles of adenine per mg of RNA sample = 𝒙 𝒙
𝟏𝟑.𝟎 𝑽𝟏 𝑾
Where:
V1= Volume in ml of hydrolysate spotted on chromatogram (20 µl)
V2= total volume of supernatant (1.5 ml)
V3= ml of 0.1 N HCl used for the elution of the spot from chromatogram (2 ml)
W= mg of RNA taken for hydrolysis (80 mg)
73
Restriction Enzyme digestion of DNA molecule –DNA
fingerprints
Introduction
Restriction enzymes are proteins that cut double stranded DNA at specific
recognition sites. Restriction enzymes are isolated from bacteria. The bacteria
use them as protection against the invasion of foreign DNA. This recognition site
or sequence is generally from 4 to 6 base pairs in length. A striking characteristic
of these cleavage sites is that they almost always possess twofold rotational
symmetry. In other words, the recognized sequence is palindromic, and the
cleavage sites are symmetrically positioned. In each strand, the enzyme cleaves
the C-G phosphodiester bond on the 3 side of the symmetry axis. Palindromes
are groups of letters that read the same in both the forward and backwards
orientation.
Types of restriction enzyme system:
Type I enzymes: Type I restriction enzymes exhibit both restriction and DNA
modification activities. They require the cofactors such as Mg2+ ions, S-
adenosylmethionine (SAM) and ATP for their activity. The recognition sequences
are quite long with no recognizable features such as symmetry. Type I restriction
endo nucleases cleaves DNA at nonspecific sites and that can be 1000 base pair
or more from recognition sequence.
Type II enzymes: Type II enzymes and their corresponding modification

methyltransferases act as separate proteins. They have a number of advantages
over type I and III systems. First, restriction and modiﬁcation are mediated by
74
separate enzymes so it is possible to cleave DNA in the absence of modiﬁcation.
Secondly, the restriction activities do not require cofactors such as ATP or S-
adenosylmethionine, making them easier to use. They require only Mg2+ ions as
cofactors. These enzymes are site-specific as they hydrolyze specific
phosphodiester bonds in both DNA strands. Class II restriction endonucleases
are generally used as the key material in molecular biology and recombinant
DNA techniques, including genome mapping, RFLP analysis, DNA sequencing,
and cloning.
Type III enzymes: Like Class I enzymes, Type III enzymes possess both
restriction and modification activities. They recognize specific sequences
and cleave 25 - 27 base pairs outside of the recognition sequence, in a 3´
direction. They require Mg2+ ions for their activity.
The names of restriction enzymes consist of a three-letter abbreviation for the

host organism (e.g., Eco for Escherichia coli, Hin for Haemophilus influenzae,
Hae for Haemophilus aegyptius) followed by a strain designation (if needed) and
a roman numeral (if more than one restriction enzyme from the same strain has
been identified). A piece of DNA produced by the action of one restriction
enzyme can be specifically cleaved into smaller fragments by another restriction
enzyme. The pattern of such fragments can serve as a fingerprint of a DNA
molecule.
The cut generated by these enzymes is either even cut or a staggered cut. The
staggered cuts made by this enzyme produce complementary single-stranded
75
ends, which have specific affinity for each other and hence are known as
cohesive or sticky
ends.
In this experiment, we will use restriction enzymes to cut up DNA from a small
virus called Bacteriophage λ. This virus is 48,502 base pairs in length which is
very small compared with the human genome of approximately 3 billion base
pairs. Since the whole sequence of λ is already known we can predict where
each restriction enzyme will cut and thus the expected size of the fragments that
will be produced. This will result in fragments with different sizes. This is termed
a partial restriction digestion.
In this experiment, we will perform

a full restriction digestion. After
overnight digestion, the reaction is
stopped by addition of a loading
buffer. The DNA fragments are
separated by electrophoresis. The
gel is then stained with a
methylene blue stain to visualize
the DNA bands and may be
photographed. Following staining to locate the DNA, the gel is observed and the
fragments appear as a pattern of bands. In this experiment, we will compare our
banding pattern with a predicted result shown in figure 1.
76
This laboratory will take approximately 3 days. The restriction digestion takes
place overnight and can be kept in the freezer until the next class period when it
will be be used for gel electrophoresis. The gels may be stained overnight prior to
photographing or recording results.
Objectives
1. Understand what a DNA restriction enzyme is and how it works.

2. Understand how to use a restriction digestion map to identify a sample
DNA.
3. Compare the λ DNA bands on a gel to the known λ DNA restriction map.
Student Activity : Restriction Enzyme Analysis
Procedure
1. Put on gloves. Keep all enzyme and DNA aliquots on ice through step 6.
2. Label 4 microtubes, reagents as indicated below, and place them in the
tube rack:
Reagents BamHI EcoRI HindIII Control
10X restriction enzyme

4 µl 4 µl 4 µl 4 µl
buffer
DNA 4 µl 4 µl 4 µl 4 µl
BamHI 2 µl 0 0 0
EcoRI 0 2 µl 0 0
HindIII 0 0 2 µl 0
Water 30µl 30µl 30µl 32µl
77
3. Carefully add 4 µl of 10X restriction buffer to each tube. Then add 4µl of
DNA to each tube, using a new tip each time.
4. Add 32µl of distilled water to the control tube and 30µl to the other reaction
tubes.
5. Close the microtubes and heat in a 55°C water bath for 10 minutes then
immediately place on ice for 2 minutes.
6. Add 2 µl of the appropriate restriction enzyme to the reaction tubes as

indicated on the grid. Use a new tip for each enzyme added.
7. Close the microtube caps and make sure that all the liquid is at the bottom
of the tube by tapping the bottom of the tube gently on the desk top. Give
the tubes to the instructor. They will be incubated at 37°C overnight.
8. Run gel Electrophoresis for about 30-45 minutes.
9. Place gel in a 0.002% methylene blue solution in 0.1X TBE and stain
overnight at 4°C or for 2 hours at room temperature. Or stain the gel with
ethidium bromide. Photograph if desired.
Student Activity
Restriction enzymes cut at specific sites along the DNA. These sites are
determined by the sequence of bases which usually form palindromes.
Palindromes are groups of letters that read the same in both the forward and
backwards orientation. In the case of DNA the letters are found on both the
forward and the reverse strands of the DNA. For example, the 5’ to 3’ strand may
have the sequence GAATTC. The complimentary bases on the opposite strand
will be CTTAAG, which is the same as reading the first strand backwards! Many
enzymes recognize these types of sequences and will attach to the DNA at this
site and then cut the strand between two of the bases. The restriction enzymes
78
which we used in this laboratory are EcoRI, HindIII and BamHI and their
sequences are as follows, with the cut site indicated by the arrow.
5'GAATTC
EcoRI
Escherichia coli 3'CTTAAG
(A)
5'GGATCC
BamHI Bacillus
3'CCTAGG\
B) amyloliquefaciens
HindIII Haemophilus 5'AAGCTT

(C) influenzae 3'TTCGAA
DNA cut with DNA cut with DNA

cut with
EcoRI(A) HindIII(C)
BamHI(B )
This figure shows the size of each of the fragments/bands produced when λ DNA
is cut with each of these restriction enzymes. The sizes were determined by
comparison to a molecular ladder which has bands of known sizes when it is
separated by electrophoresis at the same time as the digested λ DNA.
79
Restriction maps of the linear λ genome/ Restriction sites of Lambda (λ)
DNA - in base pairs (bp)
The sites at which each of the 3 different enzymes will cut lambda DNA are
shown in the maps Enzymes A, B and C below.
Lab work:
1. Calculate the size the resulting fragments will be after digestion and write a
chart containing DNA restriction fragment size.
2. How many fragments would you expect to see for each of the maps A, B
and C?
3. Compare the size of the fragments that you have calculated with the bands
shown in the photographs of the gels and determine which of the
enzymes, BamHI, EcoRI and HindIII were used to cut A, B and C. Fill the
table below with the required information?.
80
4. How many times does the sequence GAATTC occur in the λ DNA
sequence? What about AAGCTT and GGATCC?
Length of DNA The size of DNA fragments generated from digestion

fragments in bp. of DNA with
Enzyme A Enzyme B Enzyme C
50000
24000
20000
16000
10000
9000
8000
7000
6000
5000
4000
3000
2000
1000
81
DNA fingerprints
DNA fingerprinting is a laboratory technique used to establish a link between

biological evidence and a suspect in a criminal investigation. A DNA sample
taken from a crime scene is compared with a DNA sample from a suspect
(Figure 2). If the two DNA profiles are a match, then the evidence came from that
suspect. Conversely, if the two DNA profiles do not match, then the evidence
cannot have come from the suspect. DNA fingerprinting is also used to establish
paternity.
Figure. DNA finger print of the three suspects and that of victim at the crime
scene.
82
References
Aranda PS, LaJoie DM, Jorcyk C L (2012). Bleach Gel: A Simple Agarose Gel for
Analyzing RNA Quality. Electrophoresis. 33(2): 366–369. Doi: 10.1002/elps
.201100335.
Bloch KD; Grossmann B (1995). Digestion of DNA with Restriction

Endonucleases. https://doi.org/10.1002/0471142727.mb0301s31.
Chomczynski P, Sacchi N (2006). "The single-step method of RNA isolation by

acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something
years on". Nat Protoc. 1 (2): 581–5. doi:10.1038/nprot.2006.83.
Ed Rybicki (2001). PCR Primer Design and Reaction Optimization. Standard

PCR Protocol. University of Cape Town, 2001. http://www.mcb.uct.ac
.za/mcb/resources/pcr/protocol.
Elkins K M (2013). DNA Extraction Forensic DNA Biology.
Frederick M. Ausubel, Roger Brent, Robert E. Kingston, David D. Moore, J.G.

Seidman, John A. Smith, Kevin Struhl (2003). Current Protocols in Molecular
Biology. John Wiley & Sons, New York, United States.
Genetics Science Learning Center at the University of Utah. Build a Gel

Electrophoresis Chamber (2006). http://teach.
genetics.utah.edu/content/labs/build_gel_box.pdf.
Johnson M (2019). RNA extraction, Synatom Research, Princeton, New Jersey,

United States. DOI//dx.doi.org/10.13070/mm.en.2.201.
Judelson H (2012). PCR Standard Protocol (with Taq polymerase).
83
Kang G -D; Gao C-J; Chen W-D; Jie X –M; Cao Y-M; Yuan Q (2007). Study on
hypochlorite degradation of aromatic polyamide reverse osmosis membrane.
J Memb Sci. 300:165–171.
Kaufman J A. Tested laboratory procedures for disposal of small quantities of

hazardous chemicals in Waste Disposal in Academic Institutions. Lewis
Publishers, 1990, p. 127.
Lee SV (2012). Bahaman AR. Discriminatory Power of Agarose Gel

Electrophoresis in DNA Fragments Analysis. InTech. https://www.
researchgate.net/ publication/224829870
Lewis M. Agarose gel electrophoresis (basic method). Department of Pathology,

University of Liverpool. http://diyhpl.us/~bryan/irc/protocol-online/protocol-
cache/agarogel.html. Accessed at February 10, 2019.
Lunn G., Sansone E.B. Ethidium bromide: Destruction and decontamination of

solutions. Analytical Biochemistry.1987, 162, 453-458.
Madigan MT, Martinko JM. Brock TD (2006). Brock biology of microorganisms

(10th Ed). Pearson-Prentice Hall. ISBN 84-205-3679-2.
Randall DR. (2009). Molecular Biology Laboratory manual.
Sambrook JF, Russell DW (2001). Molecular Cloning: a Laboratory Manual. 3rd

edition. Cold Spring Harbor, N.Y. Cold Spring Harbor Laboratory Press.
Struhl K, Seidman J G, Moore D D, Kingston RE, Brent R, Ausubel FM,

Smith JA. (2002). Short Protocols in Molecular Biology: A Compendium of
Methods from Current Protocols in Molecular Biology. John Wiley & Sons
Inc., New York, United States.
Surzycki S (2000). basic techniques in molecular biology. Springer.
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Towbin JA (1993). Introduction to the techniques of molecular biology. Herz,
18(4):213-21.
Westermeier R (2005). Gel Electrophoresis. Encyclopedia of Life Sciences. John

Wiley & Sons. doi: 10.1038/npg.els.0005335.
Yılmaz M, Ozic C, Gok İ (2012). Principles of Nucleic Acid Separation by

Agarose Gel Electrophoresis. Gel Electrophoresis - Principles and Basics, Dr.
Magdeldin S (Ed.), ISBN: 978-953-51-0458-2, InTech. http://www.intechopen.
com/books/gel-electr ophoresis-principles-andbasics/
85
Appendix A
Safety rules and regulations in Laboratory of molecular

biology
1. At the beginning of the work in the laboratory, students have to know
laboratory safety rules and regulations in Laboratory of molecular biology.
2. It is forbidden to eat, drink and smoke in the laboratory.
3. Students have to wear laboratory coats, slippers and rubber gloves.
4. Unauthorized experiments are strictly forbidden
5. The laboratory must be kept clean and organized.
6. Check the proper installation of the equipment. If there is any problem
with equipment, do not use it and inform the supervisor.
7. Do not work with UV light on.
8. If a piece of equipment fails while being used, report it immediately to
your laboratory assistant or tutor. Never try to fix the problem yourself
because you could harm yourself or the others.
9. Clean up your work area before leaving the laboratory.
10. Turn off all electric devices before leaving the laboratory.
11. Before leaving the laboratory, wash your hands.
12. Ask the supervisor if you are in doubt.
13. Read labels carefully.
14. Never “smell” a solvent directly! Read label on the solvent bottle to
identify its contents. Chemicals must never be tasted!
15. Check where the laboratory fire extinguisher and wash station are located
and how to use them.
16. The staff and students are obliged to manipulate with poisonous, volatile
and smelly substances exclusively in running hood.
86
17. Always give the chemicals and reagents you used back to the place
where you had taken them from.
18. Special care should be taken while working with open fire, combustibles,
corrosives and toxic substances.
19. Always inform the teacher about any accident or injury and provide the
first aid if necessary.
20. The reagent solutions are always casted from the reagent bottle on the
unlabeled side to avoid the damage of the label. Illegible inscription and
incidental substitution linked with it can cause dangerous consequences.
21. Concentrated acids, especially sulphuric acid, are diluted by infusion of
acid into the water. Acid is infused in the thin stream to the solution which
is mixed up by the glass stick throughout the whole dilution.
22. Manipulation with irritating, smelling and toxic substances (i. e. chlorine,
chloroform, carbon disulphide, etc.) and easily flammable substances (i.
e. gasoline, acetone, etc.) is allowed only in well aired and functional
hood.
23. Throw toxic and nontoxic waste into the appropriate containers.
24. Everybody who work in the laboratory, have to respect all the rules
mentioned above and will inscribe their names into the presence book
Disposal and
Decontamination of Ethidium Bromide
A. Absorption Method
Dilute waste can also be decontaminated by absorption onto proprietary
absorbents which include a column marketed by Merck that changes color when
exhausted, or the Green Bag marketed by Global Scientific.
87
Waste can also be decontaminated by absorption onto Amberlite XAD-16.
Persons wishing to use these alternatives should satisfy themselves that the
products produce the desired effect when the manufacturers’ instructions are
followed.
Method for the Absorption on Amberlite XAD-16 Ion Exchange Resin – for
solutions of EtBr < 0.1 mg/ml:
• Dilute the aqueous ethidium bromide solution such that the total concentration
of ethidium bromide does not exceed 0.1 mg/mL.
• For each 100 mL aliquot of ethidium bromide solution, add approximately 3.0
grams of Amberlite XAD-16 ion exchange resin and stir the resulting mixture for
20 hours.
• Filter the Amberlite resin from the aqueous solution and place it inside a yellow
bag and send for incineration.
Activated Charcoal – for solutions of EtBr < 0.5 mg/ml

• Dilute to contain <0.5mg EtBr/ml - much liquid waste is already sufficiently
dilute (e.g. electrophoresis buffer containing 0.5mg EtBr/ml).
• Add 100mg powdered active charcoal to each 100ml solution.
• Keep at room temperature for 1 hour, shaking intermittently.
• Filter through a Whatman No. 1 filter. Discard the filtrate.
• Wrap the filter and charcoal in a plastic bag. Place inside a yellow bag and send
for incineration.
B. Bleach Decontamination Procedure Armour Method

This procedure is somewhat more complicated and is therefore only
recommended if extraction is not possible. Perform the following in a fume
hood:
• Dilute solutions of EB to a final concentration of less than or equal to 0.034%
w/v (34 mg EB/100 ml solution).
88
• Add 10 ml fresh bleach for every 1 mg EB (bleach deteriorates upon exposure
to air).
• Stir the mix continuously for 4 hours or overnight.
• Test the final solution with a UV light to ascertain that the EB is destroyed.
• Dispose final solution to sewer diluting 1 part solution with 20 parts tap water.
Please Note: this procedure is only for the decontamination of solutions

containing ethidium bromide and not for surfaces as it relies on a 4 hour
reaction time to ensure complete oxidation and conversion of all ethidium
bromide to non-mutagenic 2-carboxybenzophenone.
C. Sodium Nitrite Procedure

This procedure is somewhat more complicated and is therefore only
recommended if extraction is not possible.
• Dilute solutions with water to reduce the EtBr concentration to <0.5mg/ml.
• To the diluted solution, add 0.2 volume of fresh 5% hypophosphorous acid and
0.12 volume of fresh 0.5M sodium nitrite
IN A FUME HOOD. Mix carefully. Important: check with indicator paper that the
pH of the solution is <3.0 (if substantial amounts of buffers are present, it might
be necessary to add more hypophosphorous acid. For mixtures containing
alcohols, e.g isopropanol, 1-butanol.
• Incubate 24 hours at room temperature. (A check for loss of fluorescence can
be used to monitor completion of the inactivation process.) Add a large excess
of 1M sodium bicarbonate before discarding.
NB:
Hypophosphorous Acid - is usually supplied as a 50% solution which is corrosive
and must be handled with care. Dilute freshly before use. Sodium Nitrite -
dissolve 34.5g NaNO2 in water and dilute to 1000ml.
Note: there is a 2-fold discrepancy between the intended Molar concentration
and the instructions for making up the solution.
89
Cleaning of Equipment and Laboratory Surfaces
Contaminated with Ethidium Bromide
Glass, stainless steel, Formica, floor tiles, benches, fume hoods and the filters of
transilluminators can be successfully decontaminated using the following
technique. (No change in the optical properties of the transilluminator filter could
be detected even after a number of treatments with the decontamination
solution.)
• Unplug all electrical equipment before decontamination and wear appropriate
protective equipment, including rubber gloves, lab coat, and chemical goggles.
• Make up the decontamination solution just prior to use. Dissolve 4.2 g of
sodium nitrite in 250 ml water, IN A FUME HOOD slowly add 20 ml
hypophosphorous acid (50%) and make up to a final volume of 300 ml with
water.
• Wash the contaminated surface once with a paper towel soaked in the
decontamination solution, taking care to avoid wetting electrical components.
Then wash five times with water-soaked paper towels using a fresh towel each
time.
• Soak all the towels in decontamination solution for 1 hour before disposal by
incineration.
• Use a portable UV lamp to check that decontamination is complete. EtBr
absorbs a broad range of UV light, so short (254nm), medium (300-315nm) or
long (365-6nm) wavelength lamps can be used. Appropriate eye protection
must be worn to guard the user against UV light while the lamp is switched on.
• Neutralize the used decontamination solution with sodium bicarbonate and
discard as aqueous waste.
• Dry off the decontaminated surface or equipment. Arrange for electrical
equipment to be checked by a competent electrician before plugging in for the
first time unless you are absolutely certain that none of the electrical
90
components have been wetted. If the decontamination solution (pH 1.8) is
considered to be too corrosive for the surface to be decontaminated, then use
six H2O washes. Again, soak all towels in decontamination solution for 1 hour
before disposal.
Emergency Exposure Procedures

• If EB contacts the eyes, immediately flush them with copious amounts of cold
water for at least 15 minutes. (If it is available, an emergency eyewash is the
best and safest way to do this.)
• For skin contact, immediately wash the affected area with soap and copious
amounts of cold or cool water. If a person inhales EB dust, move him to an area
where he can breathe fresh air. After any exposure to EB (via skin, inhalation,
or eye contact), the affected person should immediately seek a medical
attention.
Appendix B
Using a Micropipette
When scientists need to accurately and precisely deliver smaller volumes of a
liquid, they use a pipette – a calibrated glass tube into which the liquid is drawn
and then released. Glass and plastic pipettes have been mainstays of chemistry
and biology laboratories for decades, and they can be relied upon to dispense
volumes down to 0.1mL.
Molecular biologists frequently use much smaller volumes of liquids in their work,
even getting down to 0.1μL (that’s one ten thousandth of a milliliter, or one ten
millionth of a liter!). For such small volumes, they need to use a micropipette.
91
Micropipettes are called a lot of different
names, most of which are based on the
companies which manufacture. For
example, you might hear them called
“Gilsons”, as a large number of these
devices used in laboratories are made
by this company. Regardless of the
manufacturer, micropipettes operate on
the same principle: a plunger is
depressed by the thumb and as it is
released, liquid is drawn into a
disposable plastic tip. When the plunger
is pressed again, the liquid is
dispensed. The tips are an important
part of the micropipette and allow the
same device to be used for different
samples (so long as you change your tip between samples) without washing.
They come in a number of different sizes and colors, depending on the
micropipette they are used with, and the volume to be dispensed.
The most commonly used tips are:

Large Blue – 200-1000μL
92
Small Yellow – 2-200μL
Small White - <2μL
They are loaded into tip boxes which are often sterilized to prevent
contamination. For this reason tip boxes should be kept closed if they are not in
use. Tips are loaded onto the end of the micropipette by pushing the end of the
device into the tip and giving two sharp taps. Once used, tips are ejected into a
sharps disposal bin using the tip eject button. Never touch the tip with your
fingers, as this poses a contamination risk.
The plunger can rest in any one of three positions: Position 1 is where the pipette
is at rest
Position 2 is reached by pushing down on the plunger until resistance is met
Position 3 is reached by pushing down from Position 2.
To Dispense Liquid:
Hold the micropipette so that the end of the tip containing tip is inside the vessel
you want to deliver it to. When delivering smaller volumes into another liquid, you
may need to put the end of the tip beneath the surface of the liquid (remember to
93
change the tip afterwards if you do this to save contaminating stock). For smaller
volumes you may also need to hold the tip against the side of the container.
Push the plunger down to Position 2. If you wish to mix two liquids together or
re-suspend a centrifuged pellet, release to Position 1 and push to Position 2 a
few times to draw up and expel the mixed liquids.
To remove the last drop of liquid from the tip, push down to Position 3. If
delivering into a liquid, remove the tip from the liquid before releasing the plunger
Release the plunger and allow it to return to Position 1.
94
Appendix C
Microcentrifuges and centrifugation
Microcentrifuge is a laboratory equipment that is driven by a motor spinning liquid

samples at extremely high speeds to separate out materials along the radial
direction at the bottom of the tube from small samples (especially of biological
material). The type of the microcentrifuge devices varies depending on the size
and sample capacity. As the name implies, microcentrifuges are smaller than
most centrifuges. 1.5 ml conical tubes (called microcentrifuge tubes), often made
of polypropylene plastic, fit into a rotor that often has 12 – 40 positions.
Principle of centrifugation
 In a solution, particles whose density is higher than that of the solvent sink
(sediment), and particles that are lighter than it float to the top.
 The greater the difference in density, the faster they move. If there is no
difference in density (isopyknic conditions), the particles stay steady.
 To take advantage of even tiny differences in density to separate various
particles in a solution, gravity can be replaced with the much more
powerful “centrifugal force” provided by a centrifuge.
 A centrifuge is a piece of equipment that puts an object in rotation around
a fixed axis (spins it in a circle), applying a potentially strong force
perpendicular to the axis of spin (outward).
 The centrifuge works using the sedimentation principle, where the
centripetal acceleration causes denser substances and particles to move
outward in the radial direction.
 At the same time, objects that are less dense are displaced and move to
the center.
95
 In a laboratory centrifuge that uses sample tubes, the radial acceleration
causes denser particles to settle to the bottom of the tube, while low-
density substances rise to the top.
Types of Microcentrifuges:
There are many different types of microcentrifuge devices. They are important in
providing accurate results in medical studies. microcentrifuge devices are used in
many different industries and medical laboratories. These microcentrifuges use
glass tubes to store the sample for the process. Although plastic tubes tend to be
less expensive. There are microcentrifuge devices that are used in blood banks
and those used in chemical laboratories and then more heavy duty ones used for
petroleum products. These include the followings:
1. Preparative microcentrifuges
96
2. Analytical microcentrifuges
3. Angle fixed microcentrifuges
4. Swing head microcentrifuges
97
5. Haematocrit microcentrifuges
How to Use a Microcentrifuge

There are a few simple rules to follow in order to ensure the safety of the micro
centrifuge device while operating it.
It is necessary to counter balance any tube placed in the microcentrifuge. This
counter balancing needs to be done according the mass and not the volume.
This is required since an unbalanced microcentrifuge can permanently damage
the device. Visual inspection of the paired samples is sufficient to establish
proper balancing.
 If an odd number of samples are being processed, use a balance tube with
an appropriate amount of water. The tube for counter balancing the device
is required to be placed in the opposite grove to the current one with the
sample.
Make the appropriate setting on the microcentrifuge device and start it. If
the sample should remain cold during the spin, there are two choices. You
98
can either use a refrigerated microcentrifuge or place a regular
microcentrifuge in a cold environment such as a cold room.
After placing the samples in the microcentrifuge, put on the rotor lid if
there is one. Lids reduce the air drag as the centrifuge spins and limit
noise. Close the lid and set the time (usually in minutes) and speed
(usually in revolutions per minute (rpm)). If you set the timer for longer
than intended, please do not force the time to a shorter time. Some
microcentrifuges can accommodate such changing of the time. Others
have broken timers and are accordingly less convenient to use. The
speed can be changed at any time.
Most microcentrifuges have a “quick spin” feature. In that case, the rotor
will spin as long as a particular button is depressed. These are usually 5 –
10 second spins.
Most microcentrifuges will automatically lock upon the spin starting. This
is a safety feature since opening the centrifuge lid during a spin may
expose the user to debris moving at high speed. After the run is complete,
a button commonly needs to be depressed to release the lock and allow
the samples to be removed.
 Make sure to let it complete the process before removing it; furthermore it
is advisable not to touch or open the device while it is in operation
99

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Laboratory Manual of

Basic Molecular Biology

Professor Dr. Najat Abdulrazzaq Hasan

In Loving Memory of my Father, Mother and Husband

To my beloved son Aws and his family

The purpose of this laboratory manual is to introduce undergraduate and

Two-to-three students per team works well to maximize peer interaction

Dr. Najat A. Hasan, 2019

General Laboratory Procedures and Safety

III. Disposal of Buffers and Chemicals

B. Operating Instructions for Spectrophotometer:

Introduction to DNA Extractions, Preparation of crude

Onion DNA Extraction

2. Add 100 ml of detergent/salt solution.

3. Blend on high 30 sec-1 minute.

5. Add 20-30 ml meat tenderizer and stir to mix.

6. Place 2 ml filtrate in a test tube.

1. Add 100 ml distilled water to a beaker and heat to 50-60°C.

2. Add 1.5 g wheat germ and mix until dissolved.

3. Add 5 ml detergent. Maintain 50-60°C temperature and stir for 5 minutes.

4. Add 3 g meat tenderizer.

5. Add baking soda solution to bring the pH to approximately 8.0.

6. Maintain the 50-60°C temperature and stir for 10 minutes.

7. Remove from heat.

8. Add 2 ml of the solution to a test tube and cool to room temperature.

Lima Bean Bacteria DNA Extraction

1. Add 14 ml of the bacterial suspension to a centrifuge tube and spin in a

3. Add 5 ml of prep buffer and re-suspend your cells with a pipette.

4. Add 1 ml 50% detergent solution.

5. Add 1 ml papaya juice.

6. Add 2 ml salt solution and shake for 2 minutes.

Yeast DNA Extraction

1. Mix 1 package of dry yeast in a beaker with 40 ml of 50 °C hot tap water to

2. Add 40 ml detergent/salt solution.

5. Place 2 ml of mixture into a test tube.

2. DNA appears fluffy which means it has sheared in the extraction

3. DNA appears as thin threads (intact DNA)

Materials and Solution preparation

 29.2 g non-iodized salt

DNA Extraction Summary Chart

What are the

What lyses the

Phenol/chloroform Extraction of DNA

1. Removal of proteins from the DNA solution depends on differences in the

The organic solvents commonly used are phenol and chloroform

2. The use of ionic detergents. These detergents, by unfolding the protein,

Concentrating the DNA:

1. Add an equal volume of buffer-saturated phenol: chloroform (1:1) to the

Ethanol Precipitation of DNA

3. Spin a 9000 rpm in a microfuge for 10min.Carefully decants the

4. Resuspend pellet in the 50 ul of sterile distilled water.

Determination of the Purity and Quantity of DNA

DNA concentration (N) = A260 / ε260……………………….(1)

N = 1 / 0.02 = 50 µg /ml ……………………………………..(2)

The absorption coefficient of single-stranded DNA is 0.027 µg/cm giving an

Reliable measurements of DNA concentration can be made for solutions of 0.5–

An absorption of 1.0 is equivalent to approximately:

50 μg/mL double-stranded DNA (dsDNA)

Absorbance measurements at wavelengths other than 260 nm are used for