Recombinant DNA Technology and Gene Manipulation
Submitted To: Dr Saba Jamil
Submitted By: Samrah Nadeem
Class: MPhil Biochemistry
Reg. No: 2021-ag-1794
This file depicts the lab safety rules, biosafety levels, and lab instruments. Further
it give an overview of the functions of chemicals and buffers in DNA extraction,
Electrophoresis and other techniques.
General lab safety rules
The following for lab safety are:
2. Ensure you are fully aware of your facility's/building's evacuation procedures.
3. Make sure you know where your lab's safety equipment how to properly use it.
4. Know emergency phone numbers to use to call for help in case of an emergency.
5. Lab areas containing carcinogens, radioisotopes, biohazards, and lasers should be properly
marked with the appropriate
7. Open flames should never be
8. Make sure you are aware of
where your lab's exits and fire
alarms are located.
9. If there is a fire drill, be sure to
turn off all electrical equipment
and close all containers.
10. Always work in properly-
11. Do not chew gum, drink, or eat
while working in the lab.
12. Laboratory glassware should never be utilized as food or beverage containers.
13. Checked the glassware every time you use to prevent any wound.
14. Never use lab equipment that you are not approved or trained by your supervisor to operate.
15. If an instrument or equipment fails during use then report immediately.
16. Being the last person to leave the lab, make sure to lock all the doors and turn off all ignition
17. Do not work alone in the lab and never leave an ongoing experiment unattended.
19. Never lift any glassware, solutions, or other types of apparatus above eye level.
20. Never smell or taste chemicals and do not pipette by mouth.
22. Make sure you always follow the proper procedures for disposing lab waste.
23. Report all injuries, accidents, and broken equipment or glass right away.
24. If you have been injured, yell out immediately and as loud as you can to ensure you get help.
25. For any chemical splashing flush the affected area with running water for at least 20 minutes.
26. If you notice any unsafe conditions in the lab, let your supervisor know as soon as possible.
It applies to laboratory work with low-risk microbes that pose little to no threat of infection. For
example, working with a non-pathogenic strain of E. coli. This laboratory setting typically
consists of research taking place on benches without the use of special contaminant equipment.
A BSL-1 lab, that require only standard microbial practices, such as:
Mechanical pipetting only (no mouth pipetting allowed)
Safe sharps handling
Avoidance of splashes or aerosols
Daily decontamination of all work surfaces when work is complete
Prohibition of food, drink and smoking materials in lab setting
Personal protective equipment, such as; eye protection, gloves and a lab coat or gown
BSL-1 labs also requires immediate decontamination after spills. Infection materials are also
decontaminated prior to disposal, generally through the use of an autoclave.
This biosafety level covers laboratories that work with agents associated with human diseases
(i.e. pathogenic or infections organisms) that pose a moderate health hazard. Examples of agents
typically worked with in a BSL-2 include equine encephalitis viruses and HIV, as well
as Staphylococcus aureus (staph infections).
. In addition to BSL 1 expectation, the following practices are required in a BSL 2 lab setting:
Appropriate personal protective equipment (PPE) must be worn, including lab coats and
gloves. Eye protection and face shields can also be worn, as needed.
All procedures that can cause infection from aerosols or splashes are performed within a
biological safety cabinet (BSC).
An autoclave or an alternative method of decontamination is available for proper
The laboratory has self-closing, lockable doors.
A sink and eyewash station should be readily available.
Biohazard warning signs
Access to a BSL-2 lab is far more restrictive than a BSL-1 lab. Outside personnel, or those with
an increased risk of contamination, are often restricted from entering when work is being
BSL-3 includes work on microbes that are either indigenous or exotic, and can cause serious or
potentially lethal disease through inhalation. Examples are yellow fever, West Nile virus, and the
bacteria that cause tuberculosis. Common requirements in a BSL-3 laboratory include:
Standard personal protective equipment must be worn, and respirators might be required
Solid-front wraparound gowns, scrub suits or coveralls are often required
All work with microbes must be performed within an appropriate BSC
Access hands-free sink and eyewash are available near the exit
Sustained directional airflow to draw air into the laboratory from clean areas towards
potentially contaminated areas (Exhaust air cannot be re-circulated)
A self-closing set of locking doors with access away from general building corridors
Access to a BSL-3 laboratory is restricted and controlled at all times.
BSL-4 labs are rare but exist in small places in the US and around the world. As the highest level
of biological safety, a BSL-4 lab consists of work with highly dangerous and exotic microbes.
Infections caused by these types of microbes are frequently fatal, and come without treatment or
vaccines. Two examples of such microbes include Ebola and Marburg viruses.
In addition to BSL-3 considerations, BSL-4 laboratories have the following containment
Personnel are required to change clothing before entering, shower upon exiting
Decontamination of all materials before exiting
Personnel must wear appropriate personal protective equipment from prior BSL levels, as
well as a full body, air-supplied, positive pressure suit
A Class III biological safety cabinet
A BSL-4 laboratory is extremely isolated often located in a separate building or in an isolated
and restricted zone of the building. The laboratory also features a dedicated supply and exhaust
air, as well as vacuum lines and decontamination systems.
Agarose Gel Electrophoresis
Agarose gel electrophoresis is one of the most common electrophoresis techniques which is
relatively simple and straightforward to perform but possesses great resolving power. The
agarose gel consists of microscopic pores that act as a molecular sieve that separates molecules
based upon the charge, size and shape.
This a powerful separation method frequently used to analyze DNA fragments generated by
restriction enzymes, and it is a convenient analytical method for separating DNA fragments of
varying sizes ranging from 100 bp to 25 kb. DNA fragments smaller than 100 bp are more
effectively separated using polyacrylamide gel electrophoresis whereas pulse-field gel
electrophoresis is used to separate DNA fragments larger than 25 kb. This technique can also
be used to separate other charged biomolecules such as RNA and proteins.
The separation medium is a gel made from agarose.
During gelation, agarose polymers associate non-covalently and form a network of
bundles whose pore sizes determine a gel’s molecular sieving properties. In general, the
higher the concentration of agarose, the smaller the pore size.
To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast
wells in the gel and a current applied.
The phosphate backbone of the DNA (and RNA) molecule is negatively charged,
therefore when placed in an electric field, DNA fragments will migrate to the positively
charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are
separated by size.
The mobility of DNA molecule is inversely proportional to gel concentration. Higher percentage
gels are sturdier (strong) and easier to handle but the mobility of molecules and staining will take
longer because of the tighter matrix of the gel. The most common agarose gel concentration for
separating dyes or DNA fragments is 0.8%. However, some experiments require agarose gels
with a higher percentage, such as 1% or 1.5%.
Size of DNA molecule
The sieving properties of the agarose gel influence the rate at which a molecule migrates. The
separation occurs because smaller molecules pass through the pores of the gel more easily than
larger ones. If the size of the two fragments is similar or identical, they will migrate together in
Different forms of DNA move through the gel at different rates; DNA molecules having a more
compact shape (e.g. plasmid DNA) moves faster through gel compared with linear DNA
fragment of the same size. The migration rate of linear fragments of DNA is inversely
proportional to the log 10 of their size in base pairs. This means that the smaller the linear
fragment, the faster it migrates through the gel.
Mobility of DNA molecule is also affected by the applied voltage. Within a range, the higher the
applied voltage, the faster the samples migrate.
Preparation of Agarose gel matrix
The centerpiece of agarose gel electrophoresis is the horizontal gel electrophoresis
apparatus. The gel is made by dissolving agarose powder in boiling buffer solution.
The concentration of agarose in a gel depends on the sizes of the DNA fragments to be separated,
with most gels ranging between 0.5%-2%. The solution is then cooled to
approximately 55°C and poured into a casting tray which serves as a mold. A well-former
template (often called a comb) is placed across the end of the casting tray to form wells when the
gel solution solidifies.
After the gel solidifies, the gel is submerged in a buffer-filled electrophoresis chamber which
contains a positive electrode (anode) at one end, and a negative electrode (cathode) at the
other. The volume of the buffer should not be greater than 1/3 of the electrophoresis chamber.
The most common gel running buffers are TAE (40 mM Tris-acetate, 1 mM EDTA) and TBE
(45 mM Tris-borate, 1 mM EDTA).
Sample preparation and loading
Samples are prepared for electrophoresis by mixing them with loading dyes. Gel loading dye is
typically made at 6X concentration (0.25% bromphenol blue, 0.25% xylene cyanol, 30%
glycerol). Loading dyes used in gel
electrophoresis serve three major purposes:
1. Add density to the sample, allowing
it to sink into the gel.
2. Provide color and simplify the
3. The dyes move at standard rates
through the gel, allowing for the
estimation of the distance that DNA
fragments have migrated.
4. These samples are delivered to the sample wells with a clean micropipette (variable
automatic micropipette is the preferred one).
Ethidium bromide can be added to the gel during this step or alternatively, the gel may also be
stained after electrophoresis in running buffer containing 0.5 μg/ml EtBr for 15-30 min, followed
by destaining in running buffer for an equal length of time.
Applying electric current and separating biomolecules
A direct current (D.C.) power source is connected to the electrophoresis apparatus and electrical
current is applied. Charged molecules in the sample enter the gel through the walls of the wells.
Molecules having a net negative charge migrate towards the positive (anode) while
net positively charged molecules migrate towards the negative (cathode). The buffer serves as
a conductor of electricity and to control the pH, which is important to the charge and stability of
biological molecules. Since DNA has a strong negative charge at neutral pH, it migrates
through the gel towards the positive electrode during electrophoresis.
The bluish-purple dye allows for visual tracking of sample migration during the electrophoresis.
The gel is run until the dye has migrated
to an appropriate distance.
The agarose gel will have to be post
stained after electrophoresis. The most
commonly used stain for visualizing DNA
is ethidium bromide (EtBr)*. Alternative
stains for DNA in agarose gels include
SYBR Gold, SYBR green, crystal violet
and methyl blue. The sensitivities of
methylene blue and crystal violet are low
compared with ethidium bromide. SYBR gold and SYBR green are highly sensitive but more
expensive than EtBr.
EtBr works by intercalating itself in the DNA molecule in a concentration-dependent manner.
When exposed to short wave ultraviolet light source (transilluminator), electrons in the aromatic
ring of the ethidium molecule are activated, which leads to the release of energy (light) as the
electrons return to ground state. This allows for an estimation of the amount of DNA in any
particular DNA band based on its intensity.
Ethidium bromide is a suspect mutagen and carcinogen so must be handled cautiously. It is a
hazardous waste so must be disposed of according to strict local and/or state guidelines. Stains
containing methylene blue are considered safer than ethidium bromide, but should still be
handled and disposed with care.
The exact sizes of separated DNA fragments can be determined by plotting the log of the
molecular weight for the different bands of a DNA standard against the distance travelled by
each band. The DNA standard contains a mixture of DNA fragments of pre-determined sizes that
can be compared against the unknown DNA samples.
DNA concentrations can be estimated by:
A. Taking absorbance at 260nm.
At 260 nm, an absorbance (A) of 1 unit corresponds to a concentration of:
50 μg/ml for dsDNA
40 μg/ml for RNA
33 μg/ml for ssDNA
20-30 µg/ml for oligonucleotides
Although this method is quick and nondestructive and gives information about the purity of the
sample (e.g., presence of protein or organic contaminants), reliable estimates are obtained only
with concentrations of at least 1 μg/ml. Additionally, this method cannot distinguish between
DNA and RNA.
B. Intensity of Ethidium Bromide Fluorescence:
The amount of DNA in a sample can be estimated from the intensity of ethidium bromide
fluorescence (fluorescence emitted by ethidium bromide is proportional to the amount of DNA).
The DNA quantity in an “unknown” solution can be estimated by comparing its level of
fluorescence with the intensity of known amounts of DNA of similar size. This method is useful
if a DNA sample is contaminated with other compounds that absorb in the UV range or is too
dilute to measure at 260 nm.
Polymerase Chain Reaction (PCR)
Polymerase chain reaction (PCR) is an efficient and cost-effective
molecular tool to copy or amplify small segments of DNA or
RNA. PCR combines the principles of complementary nucleic acid
hybridization with those of nucleic acid replication that are applied
repeatedly through numerous cycles. It results in the
exponential production of the specific target DNA/RNA sequences by
a factor of 10^7 within a relatively short period.
This in vitro amplification technique can amplify a single copy of
nucleic acid target by using two synthetic oligonucleotides “primers”
that bind to the target genomic sequence, which are extended by a Taq
polymerase (a thermostable DNA polymerase). An automated process
of repeated cycles (usually 25 to 40) of denaturation of the template
DNA (at 94°C), annealing of primers to their complementary
sequences (50°C), and primer extension (70°C) are employed for the
amplification of target sequence.
PCR was originally developed in 1983 by the American biochemist and Nobel Laureate Kary
Primer: A short segment of nucleotides, which is complementary to a section of the DNA or
RNA, which is to be amplified in the PCR. Two short DNA sequences designed to bind to the
start (forward primer) and end (reverse primer) of the target sequence is used in PCR.
Taq polymerase: A thermally stable DNA polymerase originally isolated from the thermophilic
bacterium Thermus aquaticus, which resist inactivation during denaturation temperatures and
allows primer extension at high temperature.
Components of Polymerase Chain Reactions (PCR)
DNA template (the sample DNA that contains the target sequence to amplify)
Deoxyribonucleoside triphosphates (dNTPs)
Primers (forward and reverse)
To perform PCR, the extracted sample (which contains target DNA template) is added to a tube
containing primers, free nucleotides (dNTPs), and Taq polymerase. The PCR mixture is placed
in a PCR machine. PCR machine increases and decreases the temperature of the PCR mixture in
automatic, programmed steps which generates copies of the target sequence exponentially.
Polymerase Chain Reaction (PCR) has three major steps.
1. Denaturation (strand separation): The separation of the two hydrogen-bonded
complementary chains of DNA into a pair of single-stranded polynucleotide molecules by a
process of heating (94°C to 96°C)
2. Annealing (primer binding): The temperature is lowered (45-60 °C) so the primers can attach
themselves to the single-stranded DNA strands.
3. Extension (synthesis of new DNA): It starts at the annealed primer and works its way along
the DNA strand (72°C).
Detection of PCR products
Labeled probe that is specific for the target gene sequence is used to detect PCR amplified gene
product (also known as amplicon). Based on the nature of the reporter molecule used, probe
generates radioactive, colorimetric, fluorometric, or chemiluminescent signals. Probe based
detection of amplicons serves two purposes
1. It allows visualization of the PCR product
2. It provides specificity by ensuring that the amplicon is the target sequence of interest and
not the result of non-specific amplification.
Apart from DNA based hybridization method, sometimes a simple gel electrophoresis method is
sufficient to confirm the presence of specific amplicons.
Types of polymerase chain reaction-PCR
Several modifications of PCR methods have been developed to enhance the utility of this method
in diagnostic settings based on their applications. Some of the common types of PCR are;
Real-Time PCR (quantitative PCR or qPCR)
High Fidelity PCR
Hot Start PCR
Arbitrary Primed PCR
Applications of PCR
Identification and characterization of infectious agents
Direct detection of microorganisms in patient specimens
Identification of microorganisms grown in culture
Detection of antimicrobial resistance
Investigation of strain relatedness of a pathogen of interest
Genetic fingerprinting (forensic application/paternity testing)
Detection of mutation (investigation of genetic diseases)
An autoclave is a machine that provides a physical method of sterilization by killing bacteria,
viruses, and even spores present in the material put inside of the vessel using steam under
pressure. Autoclave sterilizes the materials by heating them up to a particular temperature for a
specific period of time. The autoclave is also called a steam sterilizer that is commonly used in
healthcare facilities and industries for various purposes. The autoclave is considered a more
effective method of sterilization as it is based on moist heat sterilization.
Autoclave Principle/ Working
The autoclave works on the principle of moist heat sterilization where steam under
pressure is used to sterilize the material present inside the chamber.
The high pressure increases the boiling point of water and thus helps achieve a higher
temperature for sterilization.
Water usually boils at 100°C under normal atmospheric pressure (760 mm of Hg);
however, the boiling point of water increases if the pressure is to be increased.
Similarly, the high pressure also facilitates the rapid penetration of heat into deeper parts
of the material, and moisture present in the steam causes the coagulation of proteins
causing an irreversible loss of
function and activity of microbes.
This principle is employed in an
autoclave where the water boils at
121°C at the pressure of 15 psi or
775 mm of Hg.
When this steam comes in contact
with the surface, it kills the
microbes by giving off latent heat.
The condensed liquid ensures the
moist killing of the microbes.
Once the sterilization phase is
completed (which depends on the
level of contamination of material
inside), the pressure is released
from the inside of the chamber through the whistle.
The pressure inside the chamber is then restored back to the ambient pressure while the
components inside remain hot for some time.
Uses of Autoclave
Autoclaves are important devices to ensure the sterilization of materials containing water as they
cannot be sterilized by dry heat sterilization. Besides, autoclaves are used for various other
They are used to decontaminate specific biological waste and sterilize media,
instruments, and labware.
Regulated medical waste that might contain bacteria, viruses, and other biological
materials is recommended to be inactivated by autoclaving before disposal.
In medical labs, autoclaves are used to sterilize medical equipment, glassware, surgical
equipment, and medical wastes.
Similarly, autoclaves are used for the sterilization of culture media, autoclavable
containers, plastic tubes, and pipette tips.
The spectrophotometer is an instrument which measures an amount of light that a sample
absorbs. The spectrophotometer works by passing a light beam through a sample to measure the
light intensity of a sample.
These instruments are used in the process of measuring colour and used for monitoring colour
accuracy throughout production. They are primarily used by researchers and manufacturers
everywhere. The major Spectrophotometer Applications are limitless as they are used in
practically every industrial and commercial field. However, it finds its major applications in
liquids, plastics, paper, metals and fabrics. This helps in ensuring that the colour chosen remains
consistent from its original conception to the final, finished product.
A spectrophotometer is made up of two instruments:
The spectrometer is to produce light of any wavelength, while the photometer is to measure the
intensity of light. The spectrophotometer is designed in a way that the liquid or a sample is
placed between spectrometer and photometer. The photometer measures the amount of light that
passes through the sample and delivers a voltage signal to the display.
The spectrophotometer technique is to measure light intensity as a function of wavelength. It
does this by diffracting the light beam into a spectrum of wavelengths, detecting the intensities
with a charge-coupled device, and displaying the results as a graph on the detector and then on
the display device.
1. In the spectrophotometer, a prism (or) grating is used to split the incident beam into
2. By suitable mechanisms, waves of specific wavelengths can be manipulated to fall on the
test solution. The range of the wavelengths of the incident light can be as low as 1 to
3. The spectrophotometer is useful for measuring the absorption spectrum of a compound,
that is, the absorption of light by a solution at each wavelength.
Applications of The Spectrophotometer
Detection of concentration of substances
Detection of impurities
Structure elucidation of organic compounds
Monitoring dissolved oxygen content in freshwater and marine ecosystems
Characterization of proteins
Detection of functional groups
Respiratory gas analysis in hospitals
Molecular weight determination of compounds
The visible and UV spectrophotometer may be used to identify classes of compounds in
both the pure state and in biological preparations.
Chemicals & Buffers - Functions
Tris– DNA is pH sensitive, Tris buffer maintains the pH of the solution. Also, it interacts with
the lipopolysaccharides of the cell membrane and makes them permeable, this will help in the
lysis of the cell membrane.
EDTA– EDTA is a chelating agent and can be used to block DNase activity. DNase is an
enzyme that lyses the DNA. However, every enzyme required a cofactor to work efficiently. The
chelator EDTA blocks the activity of DNase by blocking the cofactor binding site. It will work
best in combination with Tris.
TE (Tris-EDTA) buffer system consists of Tris and EDTA and has a significant role in DNA
extraction to dissolve the DNA precipitate. DNA extraction needs a specialized buffer
system to protect the DNA from harmful chemical degradation. .
In DNA or RNA extraction, the use of EDTA readily deactivates DNase or RNase
enzymes which digest DNA or RNA, respectively. Henceforth, chelation reduces the
activities of DNase and RNase.
Tris and EDTA are important ingredients of the gel electrophoresis buffer and are used in
the preparation of TBE and TAE buffer. Again, during the electrophoresis, the gel
running buffer maintains the pH of the environment.
TE buffer is a DNA preservative that stores DNA in intact form for a longer period of
time, without degrading it..
A specialized version, high-quality TE buffer with low EDTA concentration is used for
critical downstream processing like forensic DNA analysis. Tris readily maintains the pH
while the EDTA chelates ions.
Under higher EDTA concentration the Taq DNA polymerase can’t work efficiently as it
requires the Mg2+ ions as a cofactor. Because the EDTA chelates Mg2+ and decreases
the efficiency of the Taq DNA polymerase.
The Taq DNA polymerase is an enzyme that governs DNA synthesis artificially and is
thus important for PCR analysis. To overcome this problem, we have to use the TE with
low EDTA concentration which is known as the Low TE or TE low EDTA buffer.
It has a lower buffering capacity so it can be exhausted by repeated use.
The conductivity of TAE buffer is better so dsDNA can migrate faster as compared to
DNA can be easily recovered in TAE buffer so the recovery rate of TAE buffer is higher.
TAE buffer can interfere with enzymatic reaction and protect DNA, in contrast, TBE
TAE has a lower buffering capacity while TBE has a higher buffering capacity as
compared to TAE buffer. However, the borate can react with the sugar backbone of
DNA, therefore, it is not always recommended.
If glycerol is the primary component in the DNA gel loading dye, TAE buffer is the best
choice for getting a good result.
TBE has a higher buffering capacity due to the borate.
Because of the lower conductivity, the migration of dsDNA is lower as compared with
The resolution is very good for longer DNA fragments.
Borate inhibits many DNA enzymes hence the integrity of DNA is higher in TBE buffer.
TBE buffer is a little costly and working solution requirement is higher as compared to
TAE buffer (required 1X buffer).
Borate from TBE buffer can interact with DNA so the recovery rate is lower as compared
to TAE buffer.
If glycerol is a component of your DNA gel loading dye, then TBE can be a major
setback. The borate reacts with the glycerol and decreases the activity of DNA loading
It lyses the nuclear membrane as well as a cell membrane.
It maintains the pH during the DNA extraction.
Lysis buffer maintains the integrity of the DNA (protect DNA from lysis)
It separates DNA from other cell debris.
It protects DNA from acidic degradation.
Alkaline lysis, a very common technique for purifying plasmids from bacteria, involves three
solutions. The first one contains glucose, tris-HCL buffer, EDTA, and RNAses.
The glucose creates a high solute concentration outside of the bacteria so they become a
little flabby, which makes them easier to lyse.
The EDTA works as chelating agent and tris-HCL function for buffering at pH 8
RNAse will chew up any RNA inside the cell to get it out of the way.
The second solution actually lyses the cells. This one contains SDS detergent and NaOH
It raises the pH to 12 or above, denaturing proteins inside the cell and causing DNA to
separate into single strands.
The third solution contains potassium acetate.
To restore the pH to a more neutral level so the plasmid DNA strands can come back
together. In the meantime, the denatured proteins clump up and precipitate, while the
dodecyl-sulfate ions come together with the potassium ions to form an insoluble
compound, which also precipitates from solution.
SDS (Sodium dodecyl sulfate)
Sodium dodecyl sulfate is an anionic detergent that helps cell membranes and nuclear envelopes
to break open. The SDS removes the negative charges from the amino acid and disrupts the
confirmation of a protein. Therefore, the protein loses its structure and is stabilized by using the
NaCl (Sodium Chloride)
The Na+ ion of NaCl creates the ionic bond with the negative charge of DNA and neutralizes it.
It will help DNA comes together and protect from denaturation.
MgCl2 (Magnesium Chloride)
It protects the DNA. MgCl2 blocks the negative charge of the lipoproteins of the cell membrane.
After cell lysis, there is no compartment in the cell hence it protects DNA by mixing with other
Precipitates the protein impurities. The combination of phenol, chloroform and isoamyl alcohol
helps in the removal of protein. After centrifugation, the phenol settles in the bottom of the tube
and DNA in the aqueous phase while the denatured protein remains between both layers as a
The collected nucleic acid is precipitated with the help of chilled alcohol (isoamyl alcohol). We
can add salt as well to increase the yield of the DNA.
The main function of chloroform is to protect genomic DNA during a catastrophe. Chloroform
increases the efficiency of phenol to denature the protein. Here, chloroform allows proper
separation of the organic phase and aqueous phase and keeps DNA protected into the aqueous
phase. Also, chloroform denatures the lipid as well.
In the phenol-chloroform DNA extraction method, Isoamyl
alcohol helps in reducing foaming between interphase. It
prevents the emulsification of a solution.
The liquid phase contains DNA and the organic phase contains
lipid, proteins and other impurities. The precipitated protein
denatured and coagulated between both these phases. This will
create the cloudy, whitish- foam between interphase.
The anti-foaming agent, isoamyl alcohol stabilized the interphase
by removing the foaming and increases the purity of DNA.
It is one of the most popular indicators of DNA in agarose gel electrophoresis. Bromophenol
blue is a pH indicator. It is a weak acid and available as a light pink to a purple crystal and water-
It is even used as a color indicator, acid-base pH indicator and as a biological stain. At pH 3 it
will give a yellow color and pH above 4.8 it will give a blue color.
DNA is colorless and odorless, we can’t see its migration in a gel. Thus we need some chemicals
that can migrate above it. So that we can stop it running out of the gel. Electrophoresis
progression can be monitored by using the BPB (bromophenol blue).
DNA is less dense and hence it diffuses in a running buffer. We need to settle it on the bottom of
the well. The loading dye contains Ficoll or glycerol that gives density to the DNA sample.
Henceforth, DNA can’t come out and diffuse in the buffer.
It makes DNA settle on the bottom of the well. The settled DNA can migrates properly and gives
nice and sharpened bands on to gel.
BPB runs parallel to 100bp to 300bp in 0.8% agarose gel, 150bp in 2% agarose gel, and 50bp in
3% agarose gel concentration, so it runs ahead of the DNA fragment. Because of this, DNA
migration can be strictly monitored.
In DNA precipitation, a salt (sodium acetate) reacts with DNA. It breaks up into
. The positively charged sodium ion neutralizes negatively charged PO3
the DNA. Hydrophilic nature of DNA helps it to dissolve it in water but by reacting with sodium
acetate, DNA becomes less hydrophilic. Sodium acetate is often used with ethanol in DNA
precipitation. It neutralizes DNA by interacting with phosphates which help ethanol to
precipitate DNA. It is preferred over NaCl for its buffering capacity. The successive treatment
with 70% ethanol allows an additional purification, or wash, of the nucleic acid from the
Proteinase K is ideal for many molecular biology applications because it is able to break down
proteins and inactivate DNases and RNases that would otherwise degrade a desired sample of
DNA or RNA.
Digestion of unwanted proteins in molecular biology applications
Removal of endotoxins bound to cationic proteins such as lysozyme and RNaseA
Removal of nucleases for in situ hybridization
Prion research with respect to TSE (transmissible spongiform encephalopathies)
Isolation of genomic DNA
Isolation of cytoplasmic RNA
Isolation of highly native DNA or RNA
It is highly-suited to this application since the enzyme is active in the presence of chemicals that
denature proteins, such as SDS and urea, chelating agents such as EDTA, sulfhydryl reagents, as
well as trypsin or chymotrypsin inhibitors. Proteinase K is used for the destruction of proteins in
cell lysates (tissue, cell culture cells) and for the release of nucleic acids, since it very effectively
inactivates DNases and RNases.
DNases and RNases
DNases or RNases are enzymes capable of degrading DNA or RNA by catalyzing the hydrolytic
cleavage of phosphodiester bonds in the DNA or RNA backbone. These enzymes are distributed
everywhere in the body and play vital roles in maintaining the normal function of the body.
CTAB (also called hexadecyl-trimethylammonium bromide) is a cationic detergent
that facilitates the separation of polysaccharides during purification while additives, such as
polyvinyl pyrrolidone, aid in inactivating polyphenols. CTAB based extraction buffers are
widely used when purifying DNA from plant tissues.
This detergent solubilizes the plant cell wall and lipid membranes of internal organelles. Also it
denatures proteins. Thus DNA is not hydrolyzed during the isolation process. It disrupt the
membrane by releasing the DNA. Further CTAB along with other chemicals like PVP protects
the DNA from metabolites like polyphenols etc.
Polyvinyl pyrrolidone (PVP)
PVP is the main ingredient along with our CTAB. Polyphenolic compounds are naturally
occurring in the plants, their major constituents are phenolic rings, but they are secreted during
the tissue damage. The polyphenolic compounds are the major inhibitors of PCR reaction.
The polyvinyl pyrrolidone (PVP) binds to the phenolic ring of tannins and prevents it’s
interaction with DNA and protects it.
It plays a supporting role in the CTAB method. It is used as an antioxidant which prevents
oxidation of the polyphenolic compounds and helps in removing those.
DNA is less soluble in chemicals containing isopropanol then the one with ethanol. Precipitation
with ethanol requires 23 volumes while with isopropanol are 0.607 volumes. Precipitation with
isopropanol of DNA occurs at room temperature and lessens the risk of compounds like sucrose
and sodium chloride to get precipitated with it.
DNA does not dissolve in ethanol. It causes the DNA to precipitate and cold ethanol is used to
separate DNA from water based solutions. The colder temperature prevents the activity of
enzyme to denature the DNA by giving the best extraction result.
The liquid nitrogen is predominantly used in the plant DNA extraction. During the grinding, the
heat is generated which activates enzymes such as endonuclease and exonucleases. These
enzymes cleave our DNA into small pieces. Adding liquid nitrogen prevents the enzymatic
reaction. Also, the liquid nitrogen freezes tissues and helps in making a fine powder of tissues.
Liquid nitrogen helps in making the homogeneous mixture of tissues.