DNA Extraction Methods

Shahid Khan
Registration Number : 02332411025
M. Phil Pharmacology (2nd Semester)
Department of Pharmacy
Faculty of Biological Sciences
Quaid-i-Azam University
Islamabad
Contents
• Introduction
• Basic Principle
• Protocols of different methods
• Publications
• References
 Introduction
Basic Concepts, Terms and Definitions
DNA
• The polymer carries genetic instructions for
the development, functioning, growth and
reproduction of all known organisms and
many viruses.

Nucleotides
• The basic building blocks of DNA, consisting
of a nitrogenous base (adenine, thymine,
guanine, or cytosine), a sugar molecule
(deoxyribose), and a phosphate group.
Location of DNA

1. The majority of the cell's

DNA is present in the
nucleus of the eukaryotic
cell and it is called nuclear
DNA.
2. A small amount of DNA is
also present in the
mitochondria.
3. In plants, DNA is also
present in the chloroplast.
4. In prokaryotic cells, DNA is
present in the nucleoid
region in the cytoplasm.
Fig : Cell Structure of Animal, Plant and Bacterial Cell.
(Cellular and Molecular Pharmacology, First Edition, July 2021)
DNA types
Genomic DNA The complete set of DNA, including coding and non-
coding regions, present in the nucleus of eukaryotic
cells or in the nucleoid of prokaryotic cell.
Methods:Salting-Out Method, PCI etc

Mitochondrial DNA DNA located in the mitochondria, which is inherited

maternally and is separate from nuclear DNA.
Commonly extracted in studies related to genetics
and evolutionary biology.
Methods: Differential Centrifugation

Plasmid DNA Circular, double-stranded DNA found in bacteria and

some other cells, often used in genetic engineering
and cloning.
Methods:Alkaline Lysis Method
Fig : Genome Organization.
(Cellular and Molecular Pharmacology, First Edition, July 2021)
Definition of DNA Extraction
• Separation of the DNA from the other components in a test sample. OR
• Isolation of DNA by breaking the cell membrane and nuclear membrane
with the help of chemicals, enzymes or physical disruptions is defined
as DNA extraction.
• Or simply, “Isolation of DNA from the rest of the cell components is
known as DNA extraction.”
History
First DNA Extraction Attempt (Friedrich
Miescher in 1869)
He isolated the cell material and named it the “nuclei” Later
on his student named it, a “nucleic acid”. Although Miescher
F accidentally developed a method for nucleic acid isolation,
he was not sure whether what he isolated was DNA or not.

Meselson and Stahl in 1958

They developed a full-function protocol for DNA extraction.
The density gradient centrifugation protocol was the first
protocol described for isolating DNA from E.coli bacteria.

1988 Miller et al
Proposed the use of proteinase K in DNA extraction,
nonetheless, Lahiri and Nurnberger (1991) used it
effectively along with the Nonidet P40 and SDS in
extraction
Basic Procedure
Collection of Sample

Cell Lysis

DNA stabilization
And Extraction
Precipitation

DNA Purification

Quality Analysis

Collection and storage

1. Collection of Sample
Purpose: Gather biological material containing DNA, which can be blood, tissue, or plant samples etc.

• Blood: Collected in an EDTA tube to prevent clotting and preserve cells.

• Tissue: Placed in a sterile container with a buffer solution to maintain stability.
• Plant material: Often dried or frozen to protect the DNA.
2. Cell Lysis
Purpose: Break open cell membranes to release DNA and other cell contents, classified into mechanical,
chemical, and enzymatic approaches.

Mechanical Chemical Lysis Enzymatic Lysis

Lysis
1. Bead Beating (bacterial cell 1. Detergents (such as SDS or 1. Lysozyme (peptidoglycan
lysis) Triton X-100) in bacterial cell walls)
2. Sonication 2. Osmotic Lysis 2. Proteinase K (Digests
3. Homogenization 3. Solvents like toluene or proteins)
(Often used for tissue samples chloroform. 3. Cellulase and Pectinase
and plant cells. (Used for breaking down
plant cell walls)

In some cases, a combination of methods is used to ensure efficient lysis, especially for tough cell types.
• Detergent and Enzyme Combination: SDS and lysozyme are combined to break down both cell membranes
and cell walls in certain bacteria.
• Mechanical and Chemical Lysis: Homogenization of plants followed by CTAB and Liquid Nitrogen treatment.
3. DNA Extraction and Stabilization
Stabilizing DNA is essential to prevent degradation by enzymes and other cellular components that might be
released during cell lysis.

Usually a buffer solution is formed called Extraction buffer (which contain lysis chemicals and DNA stabilizing
agents).

Chelating Agents (EDTA) Temperature Control

• EDTA is a chelating agent • Lowering the temperature

that binds to divalent metal reduces the activity of
ions such as magnesium nucleases and other
(Mg²⁺) and calcium (Ca²⁺). enzymes that could degrade
• These ions are essential DNA.
cofactors for nucleases • DNA is stored at -20°C (for
(enzymes that degrade short-term) or -80°C (for
DNA). long-term preservation)
4. Precipitation
After cell lysis, will typically have a mixture containing DNA, proteins, lipids, and other cellular components.

So DNA is Precipitated from the mixture by any of the following method:

Adding Incubatio
Salt n is often incubated
• The solution
• Salt (usually sodium acetate
at -20°C to -80°C for at least 30
or sodium chloride) is added
minutes to several hours.
to the solution to neutralize
the charges on the DNA and
to facilitate its precipitation.
Adding Centrifugation
• EthanolAlcohol
or isopropanol, is
• After incubation, the solution is
added to the solution to
centrifuged at high speed
precipitate the DNA. Alcohol
(usually around 10,000-15,000
lowers the solubility of DNA
x g) for 10-15 minutes to form
in the solution. (cold
a visible pellet at the bottom of
temperature helps enhance
the tube.
DNA precipitation)
5. DNA Purification
After Precipitation, DNA purification is done to remove residual salts and other contaminants.

Alcohol is usually used followed by centrifugation for Purification purposes.

Centrifugation
Washing the DNA Pellet

• The washing process • Centrifuged at high speed

usually involves adding the (usually around 10,000
cold alcohol to the pellet, -15,000 x g) for 10-15
gently inverting the tube. minutes.

Resuspension

• Dried DNA pellet is

resuspended in an Drying the DNA Pellet
appropriate buffer like TE • Pellet is air-dried or dried in a
(for long-term storage) or
vacuum concentrator to
nuclease-free water
remove any remaining
(suitable for immediate use)
alcohol.
6. Quality Assessment
It ensures that the DNA sample is free from contaminants and is of high quality, making it suitable for downstream
applications like PCR, sequencing, or cloning.

The most common methods for DNA quality assessment include spectrophotometry, fluorometric assays, and gel
electrophoresis.

1. Spectrophotometric Analysis

• DNA absorbs light maximally at 260 nm (A260).

• Common contaminants, such as proteins, absorb maximally at 280 nm
(A280).
• A260/A280 Ratio:
 A ratio of 1.8 indicates pure DNA.
 Low Ratio (< 1.8) High Ratio (> 1.8):: Protein contamination, RNA or
presence of other contaminants.
• A260/A230 ratio:
 A pure DNA sample typically has an A260/230 ratio between 2.0 and
2.2
 2. Fluorometric Quantification

• Fluorometric assays specifically bind to DNA, offering a more accurate

DNA concentration measurement.
• Procedure:
1. A known amount of dye (Dyes such as PicoGreen, Qubit, or SYBR
Green) is mixed with the DNA sample.
2. The dye-DNA complex fluoresces upon binding, and the fluorescence
intensity is measured using a fluorometer.
3. A standard curve generated using known DNA concentrations is used
to determine the concentration of the unknown sample.

3. Agarose Gel Electrophoresis

• Gel electrophoresis is primarily used to assess the integrity and size of

DNA, providing a visual representation of the DNA quality.
• Procedure:
1. A small amount of DNA is loaded into an agarose gel and subjected to
an electric field.
2. DNA fragments migrate based on their size, with smaller fragments
Intact High-Molecular-Weight DNA:
moving faster through the gel matrix. Appears as a single, distinct band
3. After electrophoresis, the gel is stained (with a dye like ethidium Degraded DNA: Shows as a smear
bromide or GelRed) and viewed under UV light. RNA Contamination: If RNA is present, it
may appear as additional smaller bands
Storage of Extracted DNA
• DNA storage is an important aspect of DNA extraction projects.
• Following Approaches:

1. Short-Term Storage (1–2 Weeks):

• DNA can be stored at 4°C for a short period without
significant degradation.

2. Medium-Term Storage (1 Month to 1 Year):

• DNA should be stored at -20°C.

3. Long-Term Storage (Over 1 Year):

• For long-term storage should ideally be kept at -80°C
in Ultra-Low Temperature (ULT) Freezers.
Instruments for DNA Extraction
Instruments Role

1. High-speed centrifuge For centrifugation of samples

2. Waterbath Temperature Control and Dissolving DNA

3. Vortex Mixing Samples

4. UV transilluminator Result analysis

5. Agarose gel electrophoresis unit For gel electrophoresis of DNA

6. Nanodrop light spectrophotometer DNA quantification and purity check

7. Qubit fluorometer DNA quantification

8. pH meter For checking pH

9. Weigh balance Weighing the chemicals

10. Tubes, Pipettes etc. For collection and transfer of samples
Chemicals for DNA Extraction
Chemical Concentration Purpose
SDS (Sodium Dodecyl Sulfate) 0.5–2% Lyses cell membranes (The hydrophobic tail interacts with
lipids, and the hydrophilic head breaks apart membranes)
Proteinase K 100–200 Digests proteins by hydrolyzes of peptide bonds in proteins
µg/mL
EDTA 10–50 mM Inhibits DNases by chelating divalent cations (e.g., Mg²⁺ and
(Ethylenediaminetetraacetic Ca²⁺), which are essential cofactors for DNase activity
Acid)
Tris-HCl (tromethamine) 10–100 mM Maintains pH (Prevents acid hydrolysis of DNA)
Salts (NaCl etc) 100–200 mM Shields DNA charge (Stabilizes DNA, aids in precipitation and
protein aggregation)
Ethanol/Isopropanol 70% /100% DNA precipitation and washing
RNase A 10–20 µg/mL Degrades RNA
Phenol-Chloroform-Isoamyl 25:24:1 Partitions proteins into organic phase, leaving DNA in aqueous
Alcohol (PCI) phase
CTAB 1–3% Removes polysaccharides
(Cetyltrimethylammonium
Bromide)
Beta-Mercaptoethanol (BME) 0.1–2% Reduces oxidation
Setting a DNA Extraction lab
Significance of DNA Extraction
Disease Relation to DNA How DNA Extraction can help

Neurodegenerative diseases like Caused by the expended CAG DNA assist in identifying the genetic
Huntington’s Disease repeats in the HTT gene leading to mutations, guiding treatment plan,
toxic Protien production that harms interventions and monitoring etc.
neurons

Thalasemia and Sickle Cell Anemia Mutations in HBB or other related DNA assist in identifying the genetic
genes mutations, guiding treatment plan,
interventions and monitoring etc.

Breast Cancer Mutations in BRCA1/BRCA2 gene DNA assist in identifying the genetic
mutations, guiding treatment plan,
interventions and monitoring etc.
Methods to Extract DNA Physical Methods

1. Magnetic bead DNA extraction

2. Paper DNA extraction
Chemical Methods
1. Organic Methods
A) Phenol-chloroform and
isoamyl alcohol
B) CTAB DNA extraction
2. Inorganic Methods
A) Proteinase K DNA extraction
B) SDS DNA extraction
C) Salting out method
D) Silica-gel-based techniques
Differences Between Physical and Chemical Procedures

Aspect Chemical Procedures Physical Procedures

Involve the use of chemical reagents to Rely on physical processes to separate
Principle separate DNA from proteins and other DNA from cells and contaminants, often
contaminants. using mechanical or magnetic techniques.
1. Phenol-chloroform extraction 1. Magnetic bead extraction
2. CTAB extraction 2. Paper DNA extraction
3. Proteinase K DNA extraction
Examples
4. SDS extraction
5. Salting out method
6. Silica-gel-based methods
Generally more complex due to the need for Typically simpler and may require fewer
precise chemical handling and multiple steps steps, focusing on physical separation.
Complexity
for purification.
Often more time-consuming due to the Generally faster as they may involve
multiple purification steps and the need for fewer steps and less waiting time for
Time careful reagent handling. chemical reactions.
Can yield high-purity DNA suitable for While they can produce good yields, the
sensitive applications, but may involve more purity of DNA may vary based on the
Yield and Purity risks of contamination if not handled properly. method used and the sample type.
Aspect Chemical Procedures Physical Procedures
Often requires lab equipment for handling May require less specialized equipment;
Equipment Required chemicals, such as fume hoods and magnetic bead extraction can be
centrifuges. performed with simple magnets.
Chemical methods may pose safety hazards Physical methods are typically safer as
Safety due to the use of toxic reagents like phenol they do not involve hazardous chemicals.
and chloroform.
 PCI DNA Extraction Method

Introduction:
• The PCI (Phenol-Chloroform-Isoamyl Alcohol) DNA extraction method used to isolate high-quality DNA from
a variety of biological samples like animal cells, plant cells, bacteria or even plasmid.

Principle:
• The PCI method is based on the principle of differential solubility.
• In this process, phenol and chloroform are used to denature proteins, which allows
for the separation of nucleic acids from cellular components. The addition of
isoamyl alcohol reduces foaming during the extraction, facilitating a cleaner
separation.

History:
• The use of phenol and chloroform for nucleic acid extraction became prominent in the late 1970s, primarily
through the efforts of John Sambrook, E.F. Fritsch, and T. Maniatis.
 Fig : Illustration of DNA extraction by Organic method (Taken from Genetic
Education)

Advantages: Disadvantages:
• High Purity of DNA • Hazardous Chemicals
• Versatility • Complexity
• Cost-Effective • Risk of Contamination
 CTAB DNA Extraction Method

Introduction:
• The CTAB (Cetyltrimethylammonium Bromide) DNA extraction method is a popular technique used primarily
for isolating high-quality DNA from plant tissues, fungi, and some bacterial cells.

Principle:
• The CTAB method relies on the properties of cetyltrimethylammonium bromide to
disrupt cell membranes and bind to polysaccharides and proteins during the
extraction process.
• In this process, tissue is homogenized in a CTAB buffer, the detergent lyses the
cells, and the cationic CTAB forms complexes with negatively charged
polysaccharides and proteins.

History:
• In 1980, Murray and Thompson proposed an inexpensive and simple protocol for plant DNA extraction by
CTAB.
• Later on different modifications (e.g. PCI-CTAB) were done for better yield.
 Fig : Illustration of DNA extraction by CTAB Method (Taken from Genetic Education)

Advantages: Disadvantages:
• The CTAB buffers remove • Polysaccharide
polysaccharides and Contamination
Polyphenols effectively Complexity
• High Yield and Quality • Time-Consuming
• Simplicity • No versatility
• Cost-Effective
 Proteinase K DNA extraction

Introduction:
• Protein (long chain of amino acid) + ase (suffix for enzyme) + K (keratin).
• Proteinase K is a serine protease that is widely used in molecular biology for the extraction and purification of
DNA.

Principle:
• The proteolytic activity of Proteinase K
degrades the peptide bonds present in
various proteins and makes them inactive.
Thereby, it removes contaminants, cell
debris and other unwanted cellular
components from the DNA.

History:
• In 1974, Ebeling et al. discovered the serine protease- proteinase K.
• Later on recognized for their utility in DNA extraction protocols.
• It works more efficiently at temperatures 60 to 65ºC and pH 8.0.
(pH 8.0 is the most suitable for DNA extraction)

(Taken from Genetic Education)

Advantages:
• High Efficiency, Widely
accepted
• Wide Temperature Range
• Stability
• Protection Against
Nucleases

Disadvantages:
• Cost
• Potential Inhibition of
downstream applications
• Storage (stored at -4ºC and
-20ºC)
• Moderate specificity
 SDS DNA extraction

Introduction:
• The SDS (Sodium Dodecyl Sulfate) DNA extraction method is a widely used laboratory technique for isolating
DNA from various biological samples, such as cells and tissues.

Principle:
• SDS or sodium dodecyl sulfate is an anionic
detergent that digests nuclear and cell
membrane proteins.
• SDS gives a negative charge to each protein as a
function of their size. Because all of proteins have
the same shape in the gel separation they are
separated only for their size. So, SDS can be used
to aid in lysing cell during DNA extraction.

History:
• The SDS DNA extraction method gained prominence in the 1970s, largely due to advancements in molecular
biology techniques.
Advantages:
• Efficiency
• Speed
• Simplicity
• Versatility

Disadvantages:
• Contamination
• Shearing of DNA due to
Mechanical disruption
• Toxicity
 Salting Out DNA Extraction

Introduction:
• This technique is particularly popular for isolating genomic DNA from human blood samples but can be
adapted to a variety of biological tissues.

Principle:
• The salting out method exploits the principle that
high salt concentrations selectively precipitate
proteins and contaminants, leaving DNA in
solution.
• During extraction, proteins and other cellular
contaminants are precipitated by the salt, while
DNA remains in solution.

History:
• The salting-out method is a non-toxic DNA extraction method described by Miller, Dykes, and Polesky in 1988.
Advantages:
• Non-toxic reagents
• Cost-effective
• Simplicity
• High yield

Disadvantages:
• Impurities
• Limited sample type
• Lower purity for some
applications
 Silica-Based DNA Extraction Methods

Introduction:
• These are widely employed in commercially available kits.

Principle:
• The principle of silica-based DNA extraction
relies on the chemical interaction between DNA
and silica surfaces.
• In the presence of chaotropic salts (like
guanidine hydrochloride or guanidine
thiocyanate), DNA binds strongly to silica
particles or membranes.
• Once the DNA is bound to the silica, impurities
can be washed away with ethanol or isopropanol.

History:
• Silica-based DNA extraction was developed by Vogelstein and Gillespie, in 1979 who first described the
adsorption of nucleic acids to silica or glass in the presence of high concentrations of chaotropic salts.
 Types
1. Silica Spin Column Method It employs a small column filled with silica gel or silica
membrane within a centrifuge-compatible tube.

2. Silica Magnetic Bead Method Magnetic beads coated with silica are used to bind DNA.

3. Silica Resin-Based Method This method uses a resin suspension rather than a solid
membrane or bead.
4. Silica-Based Filter Plate Method Done often using 96-well plates with silica membranes or
beads embedded in the wells.
5. Silica Membrane with Vacuum Filtration Uses a vacuum manifold instead of centrifugation to pull
samples through a silica membrane.

Advantages: Disadvantages:
• High purity and yield • Costly
• Rapid and scalable • Less effective for very short
• Less risk of errors, Reliable or degraded DNA fragments
and accurate
 Magnetic Bead DNA Extraction

Introduction:
• Magnetic bead-based DNA extraction is a widely used method for purifying DNA, capitalizing on magnetic
beads coated with silica or other materials that selectively bind DNA.

Principle:
• Chaotropic agents disrupt hydrogen bonding in
water, reducing DNA solubility and promoting
binding to the bead's surface. When a magnetic
field is applied, the beads (along with the bound
DNA) are immobilized, allowing for easy washing
to remove impurities.

History:
• In the Late 1980s, Patil and Hilu explored DNA isolation
using magnetic beads.
Advantages:
• Rapid
• Simple to perform
• Can be automated
• High sensitivity, suitable for
micro DNA extraction from
forensic samples

Disadvantages:
• Impurities can effect
downstream applications.
• Costly
 Paper DNA Extraction

Introduction:
• Paper-based DNA extraction is a simplified, cost-effective approach that uses cellulose or other absorbent
paper materials to capture and preserve DNA.

Principle:
• The principle behind paper-based DNA extraction is that DNA binds
to cellulose fibers in paper, especially under conditions of high salt
concentration or heat. This binding allows for easy collection and
preservation of DNA without the need for complicated equipment.

History:
• The concept of using paper as a medium for DNA preservation and
extraction emerged in the late 20th century.
• In 2000, Whatman, Inc. filed a patent titled “FTA coated media for
use as a molecular diagnostic tool”.
Advantages:
• Cost-Effective
• Easy Storage and Transportation
• No Specialized Equipment Needed
• Rapid

Disadvantages:
• Impurities can effect downstream
applications
• Lower Purity and Yield
• Limited Sample Types
Selection of DNA Extraction Method
• Choosing the right DNA extraction method is
crucial for getting higher yields and better
purity.
• Factors affecting the selection of DNA
extraction methods:
1. Research question
2. Sample type
3. Compare the available methods
Methods DNA yield and Purity Methods DNA yield and Purity

PCI Moderate Spin-column DNA Excellent

Proteinase K DNA Good

Magnetic bead-based Excellent

Automated Excellent
• Cost and time
• Health hazards and safety
• Labor Extensive
A step-by-step guide to choosing the best DNA extraction method

1. Define your research question and determine what you have to do in your experiment.
2. Determine what type of sample you are going to process. Accordingly, search for the isolation scheme and
select methods.
3. Determine the organism from which you have to isolate the DNA.
4. Study the cellular structure and composition of the target organisms.

5. Do a comparative assessment of available DNA extraction methods based on the quality and quantity,
manpower, cost, and automation involved in the experiment.

6. Check if the method is safe to use or not.

7. Check required utilities, instruments, and chemicals are present in your lab or not

8. Ask the expert.

9. Check the method and validate it.
Basic Principle of DNA Extraction
Methods
"Removal of the cell membrane and nuclear
envelope, stabilizing DNA from DNase,
removal of cell debris, precipitation, and
washing.”

Figure : Illustration of the principle of DNA extraction. (Taken from Genetic Education)
 Aim and Purposes
• In general the aim is to separate the DNA present in the
(or nucleus of the) cell from other cellular components.
• DNA Extraction is required for a variety of molecular biologic
applications, i.e.:
1. Isolation of specific DNA from animal and plants cells for
diagnostic purpose, gene cloning.
2. To identify specific source or virulence of organisms in
community or hospital.
3. To identify the individuals like rapist, war victims, thieves,
paternity identification.
Protocols
Stepwise guidance of Methods
 Protocols for DNA EXTRACTION by PCI Method

Materials

1. Microcentrifuge (capable of up to 14,000

1. Lysis Buffer (e.g., 50 mM Tris-HCl, 10 mM rpm)
EDTA, 100 mM NaCl, 1% SDS) 2. Pipettes and Sterile Tips
2. Proteinase K (20 mg/mL stock solution) 3. Vortex Mixer
3. PCI Solution (25:24:1 mixture of phenol, 4. Water Bath(37°C and 55°C)
chloroform, and isoamyl alcohol) 5. Spectrophotometer or NanoDrop (for
4. Absolute Ethanol (chilled at -20°C) or DNA quantification)
isopropanol (chilled at -20°C) 6. Sterile 1.5 mL and 2 mL Microcentrifuge
5. 70% Ethanol (prepared fresh for washing) Tubes
6. Sodium Acetate (3M, pH 5.2) or NaCl (5M 7. Ethanol-safe storage tubes (for long-
solution) term DNA storage)
7. TE Buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0)
or nuclease-free water
 Procedure

1. Sample Preparation
Animals

Tissue Samples:
Use approximately 20–30 mg of tissue and finely mince it.
Transfer to a 1.5 mL microcentrifuge tube.

Cell Culture:
Collect approximately 1x10^6 cells and centrifuge at 3,000 Plants
rpm for 5 minutes at room temperature to pellet the cells.
Discard the supernatant.

Plasmid Bacteria Fungi

 2.Cell Lysis

1. Add Lysis Buffer: Add 500 µL of lysis buffer to the sample

and mix well.
2. Add Proteinase K: Add 10–20 µL of Proteinase K (20 mg/mL
stock)
3. Incubate: Place the tube in a water bath or heat block at 55°C
for 30–60 minutes to allow for complete lysis. Briefly vortex the
sample every 15 minutes to ensure mixing.

3 Extraction

1. Add PCI Solution: Add an equal volume (500 µL) of the PCI solution to the
lysate in the tube. To improve purity,
2. Mixing : Vortex briefly (5–10 seconds) or invert the tube gently multiple times repeat the steps
to mix. 1,2,3 on the
3. Centrifuge: Place the tube in a microcentrifuge and spin at 12,000 rpm for 10 collected Aquas
minutes at room temperature. solution
4. Collecting Aquas layer: After centrifugation, carefully pipette the upper
aqueous phase (~500 µL) containing DNA into a fresh tube.
 4 DNA Precipitation

1. Add Cold Ethanol or Isopropanol: To the extracted aqueous phase, add 2–2.5
volumes (about 1 mL) of cold absolute ethanol or 1 volume (500 µL) of isopropanol.
2. Add Salt: For improved precipitation, add 0.1 volume (50 µL) of 3M sodium acetate
(pH 5.2) or 5M NaCl solution.
3. Mix and Incubate: Invert the tube gently to mix and incubate at -20°C for at least 30
minutes (or overnight for maximum yield).

5. DNA Purification

1. Centrifuge to Pellet DNA: Spin the tube at 14,000 rpm for 15 minutes at 4°C to
pellet the DNA (at the bottom of tube).
2. Remove Supernatant: Carefully discard the supernatant without disturbing the
DNA pellet.
3. Wash with 70% Ethanol: Add 500 µL of 70% ethanol, gently invert to wash the
pellet, and centrifuge at 10,000 rpm for 5 minutes at 4°C.
4. Air Dry: After removal of supernatant, allow the DNA pellet to air-dry for 5–10
minutes.
 5. Collection, Quality Analysis and Storage

1. Resuspend DNA: Add 50–100 µL of TE buffer or nuclease-free water to the dried

DNA pellet and incubate at 37°C for 10 minutes to ensure full resuspension.
2. Quantification and Purity Check: Measure DNA concentration and purity using a
spectrophotometer or NanoDrop. A 260/280 ratio between 1.8 indicates good purity.
3. Storage: Store the DNA solution at -20°C or -80°C for long-term storage.
 Protocols for DNA EXTRACTION by CTAB method

Materials

1. Microcentrifuge (capable of up to 30000

1. CTAB Extraction Buffer: (2% CTAB, 100 mM rpm)
Tris-HCl, 20 mM EDTA, 1.4 M NaCl, 2% 2. Pipettes and Sterile Tips (various
polyvinylpyrrolidone (PVP), pH 8.0) volumes)
2. Liquid nitrogen 3. Vortex Mixer
3. Proteinase K (optional, 20 mg/mL stock 4. Water Bath or Heat Block (37°C and 55°C)
solution) 5. Spectrophotometer or NanoDrop (for
4. Chloroform: Isoamyl Alcohol (24:1) (Optional) DNA quantification)
5. Isopropanol (cold, for DNA precipitation) 6. Sterile 1.5 mL and 2 mL Microcentrifuge
6. 70% Ethanol (for washing) Tubes
7. RNase A (optional, 10 mg/mL stock solution) 7. Ethanol-safe storage tubes (for long-
8. TE Buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) term DNA storage)
or nuclease-free water for DNA resuspension
 Procedure

1. Sample Preparation 2. Cell Lysis

Plant Tissue: Start with 50–100 mg of fresh or 1. Add CTAB Extraction Buffer: Add 700 µL of pre-
frozen plant tissue. Grind the tissue thoroughly warmed (65°C) CTAB extraction buffer to the
in liquid nitrogen using a mortar and pestle powdered tissue.
until a fine powder is achieved. (If High Protien content, we can use Proteinase K)
2. Mix and Incubate: Mix the contents by inverting the
Transfer Sample: Place the powdered tissue in tube and incubate at 65°C for 30–60 minutes in a
a sterile 1.5 mL microcentrifuge tube. water bath. Vortex briefly every 10–15 minutes to
ensure proper mixing.
 3. DNA Extraction

1. Add Chloroform: Isoamyl Alcohol: Add an equal volume (approximately 700 µL) of chloroform:isoamyl alcohol
(24:1) to the lysate and Mix the Phases by Vortex the mixture for 5–10 seconds or invert the tube multiple
times until emulsified.
2. OR alternatively we can Boil the sample for 30 minutes at 60°C to 65°C.
3. Centrifuge the sample at 25,000rpm (if was boiled) for 5 to 8 minutes or Centrifuge at 12,000 rpm (around
14,000 g) for 10 minutes at room temperature (if CI used) and transfer the supernatant into another tube.
 4. Precipitation

1. Add Cold Isopropanol: Add 0.6–1 volume of cold isopropanol (approximately 400–700 µL) to the aqueous
phase.
2. Add Salt (if needed): Optionally, add 0.1 volume of 5M NaCl to enhance DNA precipitation, especially in
samples with high polysaccharide content.
3. Mix and Incubate: Gently mix by inverting, then incubate the tube at -20°C for 30 minutes (or overnight for
maximum yield).
4. Centrifuge to Pellet DNA: Spin at 14,000 rpm for 15 minutes at 4°C to pellet the DNA.
 5. DNA Purification

1. Wash with 70% Ethanol: Carefully discard the supernatant, add 500 µL of 70% ethanol to the DNA pellet, and
gently invert to wash away residual salts and impurities.
2. Centrifuge again at 10,000 rpm for 5 minutes and Discard the supernatant.
3. Air-Dry: Then air-dry the DNA pellet for 5–10 minutes, ensuring it is free from ethanol but not overdried.

Discard the
supernatant
 6. Collection, Quality Analysis and Storage

1. Resuspend DNA: Add 50–100 µL of TE buffer or nuclease-free water to the dried DNA pellet and incubate at 37
°C for 10 minutes to ensure full resuspension.
2. Quantification and Purity Check: Measure DNA concentration and purity using a spectrophotometer or
NanoDrop. A 260/280 ratio between 1.8 indicates good purity.
3. Storage: Store the DNA solution at -20°C or -80°C for long-term storage.
 Protocols for DNA EXTRACTION by SDS-Proteinase K method

Materials

1. Microcentrifuge (capable of up to 14,000

1. Lysis Buffer: (e.g., 50 mM Tris-HCl, 10 mM rpm)
EDTA, 100 mM NaCl, 1% SDS; pH 8.0) 2. Pipettes and Sterile Tips (various
2. Proteinase K (20 mg/mL stock solution) volumes)
3. RNase A (optional, 10 mg/mL stock solution, 3. Vortex Mixer
for RNA removal) 4. Water Bath or Heat Block (37°C and 55°C)
4. Phenol: Chloroform: Isoamyl Alcohol (optional, 5. Spectrophotometer or NanoDrop (for
for improved purity) DNA quantification)
5. Isopropanol or Ethanol (for DNA precipitation) 6. Sterile 1.5 mL and 2 mL Microcentrifuge
6. 70% Ethanol (for washing) Tubes
7. TE Buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) 7. Ethanol-safe storage tubes (for long-
or nuclease-free water (for DNA resuspension) term DNA storage)
 Procedure

1. Sample Preparation 2. Cell Lysis

Tissue Samples: Use 20–30 mg of animal or 1. Add Lysis Buffer: Add 500 µL of lysis buffer to the
plant tissue and grind it in a mortar and pestle sample.
if necessary. Place in a sterile 1.5 mL 2. Add Proteinase K: Add 20 µL of Proteinase K (20
microcentrifuge tube. mg/mL stock).
Cell Culture: Collect approximately 1x10^6 3. Incubate: Place the tube in a 55°C water bath or
cells by centrifuging at 3,000 rpm for 5 heat block for 1–3 hours (overnight if needed),
minutes, then discard the supernatant. allowing Proteinase K to digest the proteins and
lyse the cells. Vortex briefly every 20–30 minutes
for even mixing.
 3. DNA Extraction
RNA Removal (Optional)
1. Add RNase A: If RNA contamination is a
Centrifuge: Spin at 12,000 rpm (around
concern, add 5–10 µL of RNase A (10
14,000 g) for 10 minutes at room
mg/mL) to the lysate.
temperature.
Incubate: Mix by gentle inversion and
incubate at room temperature for 15–20
minutes.

1. Add PCI Solution (Optional) : To

But in some samples,
remove residual proteins and
we may not get the
contaminants, add an equal volume
high yield so we can
of Phenol:Chloroform:Isoamyl
add other steps here
alcohol (25:24:1) to the lysate.
like PCI 2. Vortex briefly for 5–10 seconds or
invert gently.
3. Centrifuge: Spin at 12,000 rpm for
10 minutes at room temperature.
4. Separate Layers: Carefully transfer
the upper aqueous phase to a new
tube.
 4. Precipitation

1. Add Precipitating Agent: Add an equal volume of cold isopropanol (or 2–2.5 volumes of cold absolute ethanol)
to the aqueous phase.
2. Add Salt (optional): For better precipitation, add 0.1 volume of 3M sodium acetate (pH 5.2).
3. Incubate: Gently invert the tube to mix and incubate at -20°C for 30 minutes (or longer for maximum yield).
4. Centrifuge to Pellet DNA: Spin at 14,000 rpm (approximately 16,000 g) for 10–15 minutes at 4°C to pellet the
DNA.

Alcohol and Salt

Centrifuge
 5. DNA Purification

1. Wash with 70% Ethanol: Carefully discard the supernatant, add 500 µL of 70% ethanol to the DNA pellet, and
gently invert to wash away residual salts and impurities.
2. Centrifuge again at 10,000 rpm (around 12,000 g) for 5 minutes and Discard the supernatant.
3. Air-Dry: Then air-dry the DNA pellet for 5–10 minutes, ensuring it is free from ethanol but not overdried.

Discard the
supernatant
 6. Collection, Quality Analysis and Storage

1. Resuspend DNA: Add 50–100 µL of TE buffer or nuclease-free water to the dried DNA pellet and incubate at 37
°C for 10 minutes to ensure full resuspension.
2. Quantification and Purity Check: Measure DNA concentration and purity using a spectrophotometer or
NanoDrop. A 260/280 ratio between 1.8 indicates good purity.
3. Storage: Store the DNA solution at -20°C or -80°C for long-term storage.
 Protocols for DNA EXTRACTION by Salting Out method

Materials

1. Salting-Out Buffer: (e.g., 50 mM Tris-HCl, 10 1. Microcentrifuge (capable of up to 14,000 rpm)

mM EDTA, 100 mM NaCl, 1% SDS, pH 8.0) 2. Pipettes and Sterile Tips (various volumes)
2. Proteinase K (20 mg/mL stock solution, 3. Vortex Mixer
optional for enhanced protein digestion) 4. Water Bath or Heat Block (37°C and 55°C)
3. Saturated NaCl Solution (typically 6M, prepared 5. Spectrophotometer or NanoDrop (for DNA
fresh) quantification)
4. Isopropanol (cold, for DNA precipitation) 6. Sterile 1.5 mL and 2 mL Microcentrifuge
5. 70% Ethanol (for washing) Tubes
6. TE Buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) 7. Ethanol-safe storage tubes (for long-term
or nuclease-free water (for DNA resuspension) DNA storage)
 Procedure

1. Sample Preparation 2. Cell Lysis

Blood Samples: Use 100–200 µL of fresh or 1. Add Lysis Buffer: Add 500 µL of lysis buffer to the
stored blood. sample.
2. Add Proteinase K: Add 20 µL of Proteinase K (20
Tissue Samples: Use approximately 20–30 mg mg/mL stock).
of fresh tissue. If using tissue, grind to a fine 3. Incubate: Place the tube in a 55°C water bath or
powder using liquid nitrogen and place in a 1.5 heat block for 1–3 hours (overnight if needed),
mL microcentrifuge tube. allowing Proteinase K to digest the proteins and
lyse the cells. Vortex briefly every 20–30 minutes
for even mixing.
 3. DNA Extraction

1. Add Saturated NaCl Solution: Add 300 µL of saturated NaCl solution (6M)
to the lysate.
2. Mix and Vortex: Vortex the tube vigorously for 10–20 seconds to
thoroughly mix the NaCl solution with the lysate, which will precipitate
proteins and other contaminants.
3. Centrifuge: Centrifuge at 12,000 rpm (around 14,000 g) for 10 minutes at
room temperature. After centrifugation, a white pellet of precipitated
proteins should form at the bottom of the tube.
4. Transfer Supernatant: Carefully transfer the clear supernatant
(containing DNA) to a new sterile microcentrifuge tube, avoiding the
protein pellet at the bottom.

White proteins pellet

 4. Precipitation

1. Add Cold Isopropanol: Add an equal volume of cold isopropanol (around 400–500 µL) to the supernatant.
2. Invert and Incubate: Gently invert the tube to mix, and incubate at -20°C for 30 minutes (or overnight for higher
yield).
3. Centrifuge: Spin at 14,000 rpm (around 16,000 g) for 10–15 minutes at 4°C to pellet the DNA.

Alcohol

Mix and incubate

 5. DNA Purification

1. Wash with 70% Ethanol: Carefully discard the supernatant, add 500 µL of 70% ethanol to the DNA pellet, and
gently invert to wash away residual salts and impurities.
2. Centrifuge again at 10,000 rpm (around 12,000 g) for 5 minutes and Discard the supernatant.
3. Air-Dry: Then air-dry the DNA pellet for 5–10 minutes, ensuring it is free from ethanol but not overdried.

Discard the
supernatant
 6. Collection, Quality Analysis and Storage

1. Resuspend DNA: Add 50–100 µL of TE buffer or nuclease-free water to the dried DNA pellet and incubate at 37
°C for 10 minutes to ensure full resuspension.
2. Quantification and Purity Check: Measure DNA concentration and purity using a spectrophotometer or
NanoDrop. A 260/280 ratio between 1.8 indicates good purity.
3. Storage: Store the DNA solution at -20°C or -80°C for long-term storage.
 Protocols for DNA EXTRACTION by Silica based techniques

Materials

1. Lysis Buffer (e.g., 50 mM Tris-HCl, 10 mM 1. Microcentrifuge (capable of

EDTA, 0.5% SDS; pH 8.0) up to 14,000 rpm)
2. Proteinase K (20 mg/mL stock solution for 2. Pipettes and Sterile Tips
tissue digestion) (various volumes)
3. Chaotropic Binding Buffer (e.g., guanidinium 3. Water Bath or Heat Block (55
thiocyanate or guanidinium hydrochloride) °C)
4. Ethanol (96–100%) (to enhance DNA binding) 4. Vortex Mixer
5. Wash Buffer 1 (e.g., 10 mM Tris-HCl, 80% 5. Spin Column with Silica
ethanol) Membrane or Silica Gel
6. Wash Buffer 2 (e.g., 10 mM Tris-HCl, 80% Beads
ethanol with a low concentration of salt) 6. Sterile 1.5 mL and 2 mL
7. Elution Buffer (e.g., 10 mM Tris-HCl or Microcentrifuge Tubes
nuclease-free water) 7. Spectrophotometer or
NanoDrop
 Procedure

1. Sample Preparation 2. Cell Lysis

Blood Samples: Use 100–200 µL of fresh or 1. Add Lysis Buffer: Add 500 µL of lysis buffer
stored blood. containing 0.5% SDS to the sample.
2. Add Proteinase K (optional): Add 20 µL of
Tissue Samples: Use approximately 20–30 mg Proteinase K (20 mg/mL) to the lysate.
of fresh tissue. If using tissue, grind to a fine 3. Incubate: Place in a 55°C water bath or heat block
powder using liquid nitrogen and place in a 1.5 for 1–2 hours, vortexing occasionally to aid cell
mL microcentrifuge tube. lysis.
 3. DNA Extraction

1. Add Binding Buffer: Add an equal volume (about 500 µL) of chaotropic binding buffer to the lysate to
disrupt hydrogen bonding in DNA.
2. Add Ethanol: Add an equal volume (500 µL) of ethanol (96–100%) to facilitate DNA binding to silica.
3. Transfer to Spin Column or Silica Beads: Transfer the mixture to a silica spin column (or add silica beads if
using a bead-based method). DNA will bind to the silica membrane or beads in the presence of the
binding buffer.
4. Centrifuge: Spin at 12,000 rpm for 1 minute, allowing DNA to bind to the silica while removing proteins and
other contaminants in the flow-through. Discard the flow-through.
 4. Washing to purify
Wash buffer 1
(Purifying buffer)
1. Wash Buffer 1: Add 500 µL of Wash Buffer 1 to the column or 10 mM Tris-HCl,
silica beads to remove any remaining proteins and salts. 80% ethanol
2. Centrifuge: Spin at 12,000 rpm for 1 minute and discard the
flow-through.
3. Wash Buffer 2: Add 500 µL of Wash Buffer 2 to remove
residual salts and other contaminants.
4. Centrifuge: Spin at 12,000 rpm for 1 minute. Discard the flow
-through and, if needed, centrifuge for an additional minute
to remove any residual ethanol. Wash buffer 2
(Purifying buffer)
10 mM Tris-HCl,
80% ethanol with a
low concentration
of salt
 5. Elution 6. Collection, Quality Analysis and Storage

1. Add Elution Buffer: Add 50–100 µL of 1. After Elution, collect eluted DNA in a new
elution buffer (e.g., 10 mM Tris-HCl) to sterile tube.
the silica membrane or beads. 2. Quantify and Assess Purity: Use a
Incubate for 1–2 minutes at room spectrophotometer or NanoDrop to
temperature to allow DNA release. measure DNA concentration and purity.
2. Centrifuge: Spin at 12,000 rpm for 1 An A260/A280 ratio between 1.8
minute to collect eluted DNA in a new generally indicates high-purity DNA.
sterile tube 3. Storage: Store the DNA at -20°C or -80°C
for long-term use.
 Protocols for DNA EXTRACTION by Magnetic bead Method

Materials

1. Lysis Buffer (e.g., 50 mM Tris-HCl, 10 mM EDTA, 1. Magnetic Rack or Magnetic Stand (for
0.5% SDS; pH 8.0) separation of magnetic beads)
2. Proteinase K (20 mg/mL stock, for tissue digestion) 2. Microcentrifuge (optional, for initial lysis)
3. Binding Buffer (e.g., guanidinium thiocyanate or 3. Pipettes
guanidinium hydrochloride, chaotropic salts to 4. Water Bath or Heat Block (55°C, for
enhance DNA binding) incubation)
4. Magnetic Beads coated with DNA-binding surface 5. Vortex Mixer
5. Ethanol (80% or higher) (for washing) 6. Sterile 1.5 mL and 2 mL Microcentrifuge
6. Wash Buffer 1 (e.g., 80% ethanol in Tris-HCl buffer) Tubes
7. Wash Buffer 2 (e.g., 80% ethanol with low salt 7. Spectrophotometer or NanoDrop (for
content) DNA quantification)
8. Elution Buffer (e.g., 10 mM Tris-HCl or nuclease-free
water)
 Procedure

1. Sample Preparation 2. Cell Lysis

Tissue Samples: Use about 20–30 mg of 1. Add Lysis Buffer: Add 500 µL of lysis buffer
animal or plant tissue. Grind into a fine containing 0.5% SDS to the sample.
powder using liquid nitrogen if necessary and 2. Add Proteinase K (optional): Add 20 µL of
place in a 1.5 mL microcentrifuge tube. Proteinase K (20 mg/mL) to the lysate.
3. Incubate: Place in a 55°C water bath or heat block
Blood or Cell Culture: Use 100–200 µL of for 1–2 hours, vortexing occasionally to aid cell
blood or 1x10^6 cultured cells. Centrifuge lysis.
cells at 3,000 rpm for 5 minutes to pellet and
discard the supernatant.
 3. DNA Extraction

1. Add Binding Buffer: Add an equal volume (about 500 µL) of binding buffer containing chaotropic
salts to the lysate. This buffer disrupts hydrogen bonding in DNA and promotes binding to beads.
2. Add Magnetic Beads: Add 20–50 µL of magnetic beads to the lysate, then gently vortex or invert
the tube to ensure thorough mixing.
3. Incubate and Bind: Incubate at room temperature for 5–10 minutes, allowing DNA to bind to the
surface of the magnetic beads.
4. Magnetic Separation: Place the tube on a magnetic rack for 1–2 minutes until beads are pulled to
the side, leaving the supernatant clear. Carefully discard the supernatant without disturbing the
magnetic beads.
 4. Washing to purify DNA
Wash buffer 1
(washing buffer)
We can simply wash the beads by Alcohol or can use washing 80% ethanol
buffer for good purity.
1. Wash Buffer 1: Add 500 µL of Wash Buffer 1 (80% ethanol) to
remove proteins and salts. Gently invert or vortex the tube,
then separate the beads on the magnetic rack and discard
the supernatant.
2. Wash Buffer 2: Add 500 µL of Wash Buffer 2 to further purify
DNA. Repeat the magnetic separation and discard the
supernatant.
Wash buffer 2
(Purifying buffer)
80% ethanol with a
low concentration
Washing of salt
 5. Elution 6. Quality Analysis and Storage

1. Elution Buffer: Remove residual ethanol by air 1. Quantify and Assess Purity: Use a
drying for 1–2 minutes (avoiding overdrying). spectrophotometer or NanoDrop to
Then add 50–100 µL of elution buffer (e.g., 10 measure DNA concentration and purity.
mM Tris-HCl) to resuspend the beads. An A260/A280 ratio between 1.8
2. Incubate and Elute: Incubate at room generally indicates high-purity DNA.
temperature or 55°C for 5 minutes to release 2. Storage: Store the DNA at -20°C or -80°C
DNA from the beads. for long-term use.
3. Magnetic Separation: Place the tube back on the
magnetic rack to separate the beads, then
transfer the clear eluted DNA to a new sterile
microcentrifuge tube.
 Protocols for PAPER DNA EXTRACTION

Materials

1. FTA Card or DNA-Binding Filter Paper

2. Sterile Punch, Scissors, or Blade (for removing a
sample from the card)
3. Elution Buffer (e.g., TE buffer, 10 mM Tris-HCl, or
nuclease-free water)
4. Sterile Microcentrifuge Tubes
5. Magnetic Rack (optional if using a magnetic punch)
6. Thermal Cycler or Heat Block (if heat elution is
desired)
7. Pipettes
8. Ethanol 70%
9. FTA purification reagent or wash buffer
 Procedure

1. Sample Application 2. Sample Punching

1. Apply a liquid sample, crushed tissue, or 1. Use a sterile punch, scissors, or blade to cut out a
plant leaf to the FTA card. The amount of small piece of the paper containing the sample.
sample depends on the type of sample Typically, 2–3 mm diameter punches are used for
• Liquid sample: Apply up to 40 µL each DNA extraction.
• Crushed tissue: Apply as needed 2. Place the paper disk in a sterile microcentrifuge
• Plant leaf: Apply as needed tube.

2. Allow the sample to fully absorb.

3. Air dry the card for 1–2 hours at room
temperature until completely dry. This step
stabilizes and preserves the DNA.

Storage: Store the dried card in a sealed plastic

bag with a desiccant at room temperature for
short-term storage or refrigerate for longer-term
storage.
 Repeat Step 1,2,3
3. DNA Elution two to three times 4. Quality Analysis and Storage

1. Use the eluted DNA directly for

1. Add 200-300 µL of FTA purification reagent to
downstream applications, such as PCR or
the tube containing the punched disc.
qPCR. FTA-card-eluted DNA is often
2. Incubate at room temperature for 5 minutes,
suitable for these applications without
gently inverting the tube a few times.
further purification.
3. Carefully discard the FTA purification reagent.
2. Storage of Eluted DNA: If you’re not using
4. Add 200-300 µL of 70% ethanol to the tube.
the DNA immediately, store the eluted
5. After the last wash, remove any residual
solution at -20°C.
ethanol by allowing the disc to air dry briefly or
drying with gentle heat at room temperature
for about 15-30 minutes.
6. Add 50–100 µL of elution buffer (e.g., nuclease
-free water or TE buffer) directly to the tube
containing the paper punch.
7. Incubate the disc in sterile distilled water at 95
°C for 30 minutes.
8. Optionally we can Centrifuge the tube to pellet
the disc and recover condensation.
9. Carefully remove the paper punch from the
solution with sterile forceps or a pipette tip.
Publication
Article about DNA Extraction
1. Procedure of DNA Extraction
from Buccal Mucosa

(Self)
• The importance of DNA and its
structure and function are central to the
UK science curriculum at every level of
education from the age of 12 upward.
• The International Centre for Life
Science offers DNA extraction
workshops to school groups to promote
interest in science and Genomic studies.
• Students extracted DNA using a ‘‘Genes
in a bottle kit’’ (Bio-Rad,UK) which
allows students to extract, prepare, and
precipitate their own DNA from buccal
mucosa.
Criteria for optimum DNA Extraction Protocols

1. Safety: The protocol must be appropriate for use.

2. DNA yield: The main objective of the DNA workshop is to produce an easily visible
quantity of DNA. But can be quantified by certain techniques.
3. DNA quality/safety: The appearance of precipitated DNA is stereotypically
characterized as ‘‘white fluffy strands’’
4. Cost: The cost of running DNA extraction experiments can be relatively high,
prohibiting the regular use of the practical in educational settings. The primary
purpose of this research is to minimize the cost per participant, allowing wide use of
the protocol.
5. User friendliness: The protocol should be easy tofollow and quick to carry out as this
allows more time to explain the science behind each step. The protocol should be
easily carried out without any previous knowledge of practical laboratory techniques.
6. Time: This is the biggest constraint since the workshop must run in 1 hr., including
time to explain background to the practical as well as the practical itself. The protocol
is only feasible if it can be conducted in approximately 30 min as this allows the
required time for teaching and logistics.
Requirements:
1. 10 mL Lucozade Hydro Fitness Active Water
2. 1 lolly stick
3. 1 beaker or cup
4. 5 mL pipette
5. 1 micro centrifuge tube
6. 1 mL buffer: TE (10 mM Tris-HCL, 1 mM EDTA, pH 8) +
1% Sodium Dodecyl Sulphate (SDS)
7. 20 µL Proteinase K (20 mg/mL; activity>600 mAU/mL)
8. 100 µL 2.5 M Sodium Chloride
9. 5 mL acrylic test tube
10. 1 mL absolute ethanol at freezer temperature (will also
require the use of a microcentrifuge, vortex and a water
bath at 56 centigrade)
 Protocols:

1. Dispense 10 mL of Lucozade HAFW into a drinking cup.

2. Gently scrape the mucosa with a wooden lolly stick for 30
seconds. Avoid swallowing.
3. Harvest the cells by swishing the mouthwash around the mouth
1. Cell Harvesting
constantly for 60 sec;simultaneously massage the cheeks against
the mouth to increase the yield of cells.
4. Expectorate the solution back into the cup.
 1. Whilst mixing the solution, pipette 1.5 mL of the cell suspension
into a microtube.
2. Centrifuge at 10,000 rpm for 30 sec.
3. Pour off the supernatant, leaving behind a small amount of liquid
to avoid losing the cell pellet.
2.Cell Lysis
4. Repeat steps 1–3 of the cell lysis stage, adding more cell
solution to the tube each time to increase the cell pellet.
5. Add 1 mL of lysis buffer [TE pH 8 + 1% SDS]
6. Vortex for approximately 30 sec or until mixed.
7. Add 20 lL of Proteinase K.
8. Incubate the lysate at 56 centigrade for 10 min.
 1. Add 100 µL of 2.5 M sodium chloride.
2. Mix gently by inverting the tube five times.
3. Transfer to a 5 mL acrylic test tube.
4. Slowly pipette approximately 1 mL (an equal
volume to that of the lysate) of ice cold absolute
ethanol down the side of the tube whilst holding it
3. DNA precipitation
at 45 angle so that it forms layer on top of the
aqueous layer.
5. Allow the tube to stand undisturbed for 5 min
at room temperature.
6. One may also encourage precipitation at the
interface by briskly shaking the tube.
References
• Molecular pharmacology, DNA to Drug Discovery, Wiley-Blackwell; 1st
edition (January 22, 2013).
• Genetic Education Inc. (https://geneticeducation.co.in/10-different-
types-of-dna-extraction-methods-updated/)
• Cellular and Molecular Pharmacology, Pharmamed Press (February 1,
2021).
• Barbosa, C., Nogueira, S., Gadanho, M. & Chaves, S. In Molecular
Microbial Diagnostic Methods (eds Martin D’Agostino & K. Clive
Thompson) 135–154 (Academic Press, 2016).
• Dairawan, M., & Shetty, P. J. (2020). The evolution of DNA extraction
methods. American Journal of Biomedical Science and Research.
• Srivastava, Akhileshwar Kumar; Kannaujiya, Vinod Kumar; Singh, Rajesh
Kumar; Singh, Divya (5 October 2020). DNA Extraction - an overview |
ScienceDirect Topics. Elsevier Science. ISBN 978-0-12-821710-8.
• Dehasque, Marianne; Pečnerová, Patrícia; Kempe Lagerholm, Vendela;
Ersmark, Erik; Danilov, Gleb K.; Mortensen, Peter; Vartanyan, Sergey;
Dalén, Love (2022-04-13). "Development and Optimization of a Silica
Column-Based Extraction Protocol for Ancient DNA"
• Qamar W, Khan MR, Arafah A. Optimization of conditions to extract high
quality DNA for PCR analysis from whole blood using SDS-proteinase K
m ethod. S audi J Biol S ci. 2017 Nov ;24( 7):1465-1469. doi:
10.1016/j.sjbs.2016.09.016. Epub 2016 Sep 10. PMID: 30294214;
PMCID: PMC6169501.
• How Proteinase K Can be Used in Blood DNA Extraction by Sigma
Aldrich (Merck).
• Gautam, A. (2022). DNA Isolation by Lysozyme and Proteinase K. In:
DNA and RNA Isolation Techniques for Non-Experts. Techniques in Life
Science and Biomedicine for the Non-Expert. Springer, Cham. https:
//doi.org/10.1007/978-3-030-94230-4_11.
• Elkins KM (2013). "DNA Extraction". Forensic DNA Biology. pp. 39–52.
doi:10.1016/B978-0-12-394585-3.00004-3. ISBN 9780123945853.
Any Question?