1.

Phenol-Chloroform Extraction Method:

- Principle: This method is based on the differential solubility of DNA in aqueous and organic phases.
Phenol and chloroform are used to extract DNA from the sample.

- Steps:

1. Sample is lysed in a buffer containing SDS and proteinase K.

2. Phenol and chloroform are added and the sample is centrifuged.

3. DNA is present in the aqueous phase, which is separated from the organic phase.

4. DNA is precipitated using ethanol and washed with 70% ethanol.

2. Salting Out Method:

- Principle: This method is based on the fact that DNA is less soluble in high salt concentrations. Salt
is used to extract DNA from the sample.

- Steps:

1. Sample is lysed in a buffer containing SDS and proteinase K.

2. NaCl is added to the sample and the mixture is centrifuged.

3. DNA is present in the pellet, which is washed with ethanol and dried.

3. Chelex Method:

- Principle: This method uses a chelating resin (Chelex) to bind metal ions that can degrade DNA. The
resin is then removed, leaving purified DNA.

- Steps:

1. Sample is lysed in a buffer containing Chelex resin.

2. The mixture is heated to release DNA from the cells.

3. The resin is removed by centrifugation.

4. DNA is present in the supernatant and can be used directly for downstream applications.

4. CTAB Method:

- Principle: This method uses a cationic detergent (CTAB) to lyse cells and precipitate DNA.

- Steps:

1. Sample is lysed in a buffer containing CTAB and proteinase K.

2. NaCl is added to the sample and the mixture is centrifuged.

3. DNA is present in the pellet, which is washed with ethanol and dried.
5. Magnetic Bead Method:

- Principle: This method uses magnetic beads coated with a binding agent that selectively binds DNA.
The beads are then separated from the sample, leaving purified DNA.

- Steps:

1. Sample is lysed in a buffer containing magnetic beads.

2. The mixture is incubated to allow the beads to bind DNA.

3. The beads are separated from the sample using a magnetic field.

4. DNA is eluted from the beads and can be used directly for downstream applications.

6. Silica Spin Column Method:

- Principle: This method utilizes silica membranes to bind DNA, which is then washed and eluted,
resulting in purified DNA.

- Steps:

1. Sample is lysed in a buffer containing chaotropic salts and proteinase K.

2. The lysate is loaded onto a silica spin column, where DNA binds to the membrane.

3. The column is washed to remove contaminants and DNA is eluted with a low-salt buffer.

7. Organic Solvent Extraction Method:

- Principle: This method uses organic solvents such as chloroform and isoamyl alcohol to separate
DNA from other cellular components.

- Steps:

1. Sample is lysed in a buffer containing SDS and proteinase K.

2. Phenol-chloroform-isoamyl alcohol mixture is added to the sample and centrifuged.

3. DNA is present in the aqueous phase, which is separated from the organic phase.

4. DNA is precipitated using ethanol and washed with 70% ethanol.

8. Spin Column Method:

- Principle: This method uses a spin column with a silica membrane or other binding agent to
selectively bind DNA, which is then washed and eluted, resulting in purified DNA.

- Steps:

1. Sample is lysed in a buffer containing chaotropic salts and proteinase K.

2. The lysate is loaded onto the spin column, where DNA binds to the membrane.

3. The column is washed to remove contaminants and DNA is eluted with a low-salt buffer.

9. Solid Phase Extraction Method:

- Principle: This method uses a solid phase matrix to selectively bind DNA, which is then washed and
eluted, resulting in purified DNA.

- Steps:

1. Sample is lysed in a buffer containing chaotropic salts and proteinase K.

2. The lysate is loaded onto a solid phase extraction column, where DNA binds to the matrix.

3. The column is washed to remove contaminants and DNA is eluted with a low-salt buffer.

10. Immunoprecipitation Method:

- Principle: This method utilizes antibodies specific to DNA-binding proteins, allowing for the
selective isolation of DNA-protein complexes.

- Steps:

1. Sample is cross-linked to stabilize DNA-protein interactions.

2. Antibodies specific to the DNA-binding protein of interest are added to the sample, allowing for
the formation of antibody-DNA-protein complexes.

3. Protein A/G beads are added to the sample, and the complexes are immunoprecipitated.

4. DNA is then isolated from the complexes for downstream analysis.

11. Magnetic Nanoparticle-Based Method:

- Principle: This method uses functionalized magnetic nanoparticles to selectively bind DNA, which
can then be separated and eluted for downstream applications.

- Steps:

1. Sample is lysed in a buffer containing magnetic nanoparticles functionalized with DNA-binding

ligands.

2. The nanoparticles are mixed with the lysate, and DNA binds to the particles.

3. The particles are separated using a magnetic field, and DNA is eluted from the particles.

12. Microfluidic-Based Method:

- Principle: This method involves the use of microfluidic devices to manipulate and isolate DNA from
the sample using various techniques such as electrophoresis, chromatography, and solid-phase
extraction.

- Steps:

1. The sample is introduced into the microfluidic device, where DNA is selectively isolated based on
different physical and chemical properties.

2. DNA is then collected from the device for further analysis.

13. Isoelectric Focusing Method:

- Principle: This method separates DNA based on its isoelectric point using a pH gradient, allowing
for the isolation of DNA from other cellular components.

- Steps:

1. The sample is introduced into an isoelectric focusing chamber, where a pH gradient is established.

2. DNA migrates to its isoelectric point and becomes focused at a specific pH, allowing for its
isolation.

14. DNA Extraction Using Magnetic Bead-Based Automation:

- Principle: This method involves the use of automated systems that employ magnetic beads for
efficient and high-throughput DNA extraction.

- Steps:

1. The sample is introduced into the automated system, where it undergoes lysis and binding to
magnetic beads.

2. The system automates the washing and elution steps, resulting in purified DNA.

15. High-Throughput DNA Extraction Using 96-Well Plates:

- Principle: This method allows for the simultaneous extraction of DNA from multiple samples using
96-well plates, enabling high-throughput processing.

- Steps:

1. Samples are lysed and added to individual wells of a 96-well plate.

2. DNA is bound to a solid-phase matrix within each well, followed by washing and elution steps.

3. This method allows for the extraction of DNA from multiple samples in a single batch.

16. Chelex Resin-Based Method:

- Principle: This method utilizes Chelex resin, which selectively binds metal ions and allows for the
isolation of DNA from the sample.

- Steps:

1. The sample is mixed with Chelex resin in a buffer and heated to release DNA from the cells and
bind it to the resin.

2. The mixture is then centrifuged, and the supernatant containing purified DNA is collected.

17. Solid-Phase Reversible Immobilization (SPRI) Method:

- Principle: This method uses paramagnetic beads with a surface chemistry that allows for reversible
binding of DNA, enabling purification and size selection.

- Steps:

1. The sample is mixed with SPRI beads, and DNA binds to the beads in the presence of a chaotropic
salt solution.

2. The beads are then separated using a magnetic field, and DNA is eluted in a low-salt buffer.

18. Acoustic DNA Extraction:

- Principle: This method utilizes acoustic energy to selectively isolate DNA from the sample, allowing
for gentle and efficient extraction.

- Steps:

1. The sample is introduced into a chamber where acoustic waves are used to separate DNA from
other cellular components.

2. The isolated DNA is then collected for downstream analysis.

19. DNA Extraction Using Liquid-Liquid Extraction:

- Principle: This method involves the extraction of DNA by partitioning it between two immiscible
liquid phases, allowing for the isolation of DNA from the sample.

- Steps:

1. The sample is mixed with an organic solvent such as phenol-chloroform, and DNA partitions into
the organic phase.

2. The organic phase containing DNA is separated, and DNA is precipitated and washed with ethanol.

20. DNA Extraction Using Carbon Nanotubes:

- Principle: This method uses functionalized carbon nanotubes to selectively bind DNA, allowing for
efficient extraction and purification.
- Steps:

1. The sample is mixed with carbon nanotubes functionalized with DNA-binding ligands, and DNA
binds to the nanotubes.

2. The nanotubes are then separated, and DNA is eluted for downstream applications.

21. DNA Extraction Using Silica Membranes:

- Principle: This method utilizes silica membranes to selectively bind DNA, allowing for efficient
purification and elution.

- Steps:

1. The sample is passed through a silica membrane, where DNA binds to the membrane in the
presence of chaotropic salts.

2. After washing to remove contaminants, DNA is eluted from the membrane using a low-salt buffer.

22. DNA Extraction Using Spin Columns:

- Principle: This method involves the use of spin columns containing a silica-based membrane or
resin to bind DNA, facilitating purification and elution.

- Steps:

1. The sample is loaded onto the spin column, and DNA binds to the membrane while contaminants
pass through.

2. After washing to remove impurities, DNA is eluted from the column using a low-salt buffer.

23. DNA Extraction Using Organic Solvents and Salts:

- Principle: This method relies on the precipitation of DNA using organic solvents and salts, allowing
for the isolation of DNA from the sample.

- Steps:

1. The sample is mixed with an organic solvent such as ethanol or isopropanol and a high-salt buffer
to precipitate DNA.

2. The DNA precipitate is then collected by centrifugation, washed, and re-suspended in an

appropriate buffer.

24. DNA Extraction Using Filter-Based Methods:

- Principle: This method involves the use of filters or membranes with specific pore sizes to capture
DNA while allowing other components to pass through.

- Steps:
1. The sample is passed through a filter or membrane, and DNA is retained while other components
are removed.

2. DNA is then eluted from the filter or membrane for downstream applications.

25. DNA Extraction Using Lysozyme and Proteinase K Digestion:

- Principle: This method involves enzymatic digestion of the cell wall using lysozyme and subsequent
digestion of proteins using proteinase K to release and purify DNA.

- Steps:

1. The sample is treated with lysozyme to weaken the cell wall, followed by proteinase K digestion to
release DNA from the cells.

2. DNA is then purified using methods such as phenol-chloroform extraction or spin columns.

26. DNA Extraction Using Magnetic Beads:

- Principle: This method involves the use of magnetic beads functionalized with DNA-binding ligands
to isolate and purify DNA from the sample.

- Steps:

1. The sample is mixed with magnetic beads, and DNA binds to the beads in the presence of a
chaotropic salt solution.

2. The beads are then separated using a magnetic field, and DNA is eluted in a low-salt buffer.

27. DNA Extraction Using Microfluidic Devices:

- Principle: This method utilizes microfluidic devices to manipulate and process small volumes of
sample, enabling efficient DNA extraction and purification.

- Steps:

1. The sample is introduced into the microfluidic device, where it undergoes controlled fluid flow
and interactions with functionalized surfaces to isolate and purify DNA.

2. The purified DNA is then collected for downstream analysis.

28. DNA Extraction Using Anion Exchange Resins:

- Principle: This method involves the use of anion exchange resins to selectively bind DNA, enabling
purification and elution.

- Steps:

1. The sample is mixed with the anion exchange resin, and DNA binds to the resin in the presence of
a high-salt buffer.
2. After washing to remove impurities, DNA is eluted from the resin using a low-salt buffer.

29. DNA Extraction Using Differential Precipitation:

- Principle: This method utilizes differential precipitation of DNA and other cellular components,
allowing for the isolation of DNA from the sample.

- Steps:

1. The sample is treated with a reagent that selectively precipitates DNA, while other components
remain in solution.

2. The DNA precipitate is then collected by centrifugation, washed, and re-suspended in an

appropriate buffer.

30. DNA Extraction Using Microbial Cell Lysis:

- Principle: This method involves the selective lysis of microbial cells to release and purify DNA from
complex environmental samples.

- Steps:

1. Microbial cells are lysed using a combination of physical disruption, enzymatic digestion, and
chemical lysis to release DNA.

2. DNA is then purified using methods such as spin columns or organic extraction.

These methods offer a wide range of techniques for DNA extraction, each with specific advantages
and suitability for different sample types and downstream applications.
Chromatography based methods

1. Size exclusion

 Gel filtration
 Gel permeable

The terms "gel filtration" and "gel permeable chromatography" are often used interchangeably to
describe the same technique of size exclusion chromatography. There is no significant difference
between these two. Just a bit confusing because of the names.

2. Ion exchange

1. Principle of ion-exchange chromatography (IEC): IEC is a chromatographic technique used for separating biomolecules
based on their charge properties. In this method, a column is packed with an anion-exchange resin, such as DEAE cellulose,
which contains positively charged groups. When a sample containing DNA is applied to the column, the DNA, which is
negatively charged due to its phosphate backbone, will interact with the positively charged groups on the resin, leading to
its selective binding.
2. Selective binding of DNA: The DNA anion-exchange resin, with its DEAE groups, selectively binds the DNA molecules
while allowing other cellular components such as proteins, lipids, carbohydrates, metabolites, and RNA to flow through the
column. This selective binding is based on the electrostatic interactions between the negatively charged DNA and the
positively charged DEAE groups on the resin.

3. Elution of non-DNA components: After the sample has been applied to the column, the non-DNA components are eluted
using medium-salt buffers. These components do not interact strongly with the DEAE groups and are therefore able to pass
through the column, while the DNA remains bound.

4. Recovery of DNA: Once the non-DNA components have been eluted, the DNA can be recovered from the column. This
can be achieved by using a high-salt buffer or by decreasing the pH, which disrupts the electrostatic interactions between
the DNA and the DEAE groups, allowing the DNA to be released from the resin and collected for further analysis or
applications.

The counterion buffer in ion exchange chromatography serves several important purposes, and its absence would
significantly impact the separation process. Here are the key reasons for using a counterion buffer:

1. Charge Neutralization: The functional groups on the stationary phase in ion exchange chromatography are
charged. The counterion buffer contains ions with the opposite charge to the functional groups. The presence of
the counterion buffer ensures that the functional groups on the stationary phase are neutralized, preventing
non-specific binding of charged analytes to the stationary phase. Without the counterion buffer, the functional
groups would not be neutralized, leading to non-specific interactions and poor separation.

2. Competitive Binding: The counterion buffer also plays a role in competitive binding. When the sample analyte is
introduced, it competes with the counterions in the buffer to bind to the charged functional groups on the
stationary phase. This competitive binding allows for the selective retention and separation of analytes based on
their charge and affinity for the stationary phase.

3. Control of Ionic Strength and pH: The counterion buffer helps to maintain a constant ionic strength and pH in the
mobile phase, which is crucial for reproducible and controlled separations. This is particularly important for
maintaining consistent chromatographic conditions and reliable elution profiles

In ion exchange chromatography, elution of DNA from a resin such as DAEA (diethylaminoethyl) is influenced by the ionic
strength and pH of the elution buffer. Here's how high salt concentration and decreasing pH can elute DNA from DAEA
resin:

1. High Salt Concentration:

 High salt concentration in the elution buffer increases the ionic strength of the mobile phase. This high ionic
strength effectively competes with the DNA for binding to the negatively charged functional groups on the DAEA
resin.
 The high salt concentration disrupts the electrostatic interactions between the DNA and the resin, reducing the
binding affinity of the DNA for the resin.
 As a result, the DNA molecules are displaced from the resin and are eluted from the column.

2. Decreasing pH:

 Decreasing the pH of the elution buffer can also elute DNA from the DAEA resin. This is because DNA typically
carries a negative charge due to the phosphate groups in its backbone.
 At higher pH, the negatively charged DNA interacts strongly with the positively charged functional groups on the
DAEA resin, leading to strong binding and retention.
 However, as the pH is decreased, the positive charge on the functional groups of the resin is reduced, leading to
weaker electrostatic interactions with the negatively charged DNA.
 Consequently, the DNA molecules are released from the resin and are eluted from the column as the pH is
lowered.

In summary, high salt concentration and decreasing pH can elute DNA from DAEA resin in ion exchange chromatography by
disrupting the electrostatic interactions between the DNA and the resin, thereby reducing the binding affinity and leading
to the release of DNA from the resin. These elution conditions are designed to selectively release the DNA of interest while
retaining other components that may have different binding properties.

4. Affinity chromatography

The text describes the use of affinity chromatography (AC) for nucleic acid purification, specifically for the isolation of
mRNA. Affinity chromatography involves the use of ligands that form highly specific interactions with the target molecule,
in this case, nucleic acids, to achieve separation from the cell lysate.

1. Ligand Specificity: Affinity chromatography utilizes ligands such as oligo(dT) or other substances that have a high affinity
and specificity for nucleic acids, particularly mRNA. Oligo(dT) is a synthetic oligonucleotide composed of thymine bases,
which forms stable hydrogen bonding interactions with the polyadenine tail present in mRNA. This specificity allows for the
selective binding and purification of mRNA from the complex mixture of nucleic acids in the cell lysate.

2. Selective Binding: The highly specific interactions between the ligand and the target nucleic acid enable selective binding
of the mRNA to the affinity matrix. This selective binding facilitates the purification of the mRNA from other nucleic acid
species present in the cell lysate, such as rRNA and tRNA, which do not have the same affinity for the ligand.

3. Time Efficiency: Affinity chromatography for nucleic acid purification is described as time-efficient. This is because the
specific interactions between the ligand and the target nucleic acid allow for rapid and efficient capture of the mRNA from
the sample, reducing the time required for purification compared to other methods.

4. Yield of Nucleic Acid: The protocol is reported to yield a good quantity of nucleic acid, specifically mRNA. The high
specificity and efficiency of the affinity chromatography process result in a high recovery of purified mRNA, providing a
good yield of the target nucleic acid for downstream applications such as gene expression analysis and other molecular
biology studies.

In summary, affinity chromatography for nucleic acid purification, particularly for isolating mRNA, is characterized by the
use of specific ligands, selective binding, time efficiency, and high yield of the target nucleic acid, making it a valuable
method for obtaining pure and abundant mRNA from complex biological samples.
Etbr-cscl method of DNA extraction

1. Development and Pioneers: The method for DNA extraction using cesium chloride (CsCl) gradient
centrifugation was developed in 1957 by Matthew Meselson, Franklin W. Stahl, and Jerome
Vinograd. This pioneering technique revolutionized the field of molecular biology and enabled the
separation and purification of DNA based on its density.

2. Principle of Separation: In this method, DNA is mixed with a CsCl solution and subjected to ultra-
centrifugation at high speeds (10,000 to 12,000 rpm) for more than 10 hours. During centrifugation,
the DNA separates from other substances based on its density. This separation occurs as the CsCl
forms a density gradient in the centrifuge tube, allowing DNA to migrate to a specific position in the
gradient based on its density.

3. Isopycnic Point and DNA Bands: Depending on the types of DNA present, which vary in density,
one or more DNA bands appear in the CsCl gradient when the DNA reaches the isopycnic point. At
this point, the DNA bands settle at positions in the gradient corresponding to their respective
densities, allowing for their separation and visualization.

4. Use of Ethidium Bromide (EtBr): Ethidium bromide (EtBr) is incorporated into the DNA and acts as
an intercalating agent. It is comparatively more incorporated into non-supercoiled DNA molecules
than supercoiled DNA. This differential binding allows supercoiled DNA to accumulate at lower
densities in the CsCl gradient. The location of the DNA bands can be easily visualized under
ultraviolet light due to the fluorescence of the EtBr-bound DNA.

5. Removal of EtBr and CsCl: After the separation, EtBr and CsCl are removed prior to the
precipitation of DNA with ethanol, ensuring that the purified DNA is free from these chemicals.

6. Applications and Limitations: This method can be used to extract DNA from bacteria; however, it
requires a large amount of starting material. Additionally, it is considered complicated, time-
consuming, and costly due to the long duration of high-speed ultra-centrifugation required. The
need for specialized equipment and the extended centrifugation time contributes to the complexity
and cost of the method.

In summary, the CsCl gradient centrifugation method allows for the separation and purification of
DNA based on density, but its use is limited by the requirement for substantial starting material,
complexity, time consumption, and cost associated with the ultra-centrifugation process.
Alkaline extraction

1. Introduction and Pioneers: The method for extracting plasmid DNA from bacterial cells, commonly
known as the alkaline lysis method, was first introduced in 1979 by Birnboim and Doly. This
technique has become widely used for isolating plasmid DNA from bacterial cultures.

2. Alkaline Solution for Cell Lysis: The process begins by suspending the bacterial sample in an
alkaline solution containing sodium hydroxide (NaOH) and SDS detergent. The alkaline environment
serves two main purposes: it facilitates the lysis of the bacterial cell membranes and denatures the
proteins. This step effectively releases the cellular contents, including plasmid DNA, into the
solution.

3. Selective Denaturation of Chromosomal DNA: The alkaline conditions selectively denature the
chromosomal DNA, which typically has a higher molecular weight compared to the intact plasmid
DNA. The denaturation of chromosomal DNA allows it to remain single-stranded, while the plasmid
DNA, which is smaller and typically supercoiled, remains double-stranded and intact.

4. Neutralization and Precipitation: Potassium acetate is then added to the solution to neutralize the
alkaline conditions. This neutralization step causes the renaturation of the chromosomal DNA,
leading to its precipitation from the solution. Meanwhile, the plasmid DNA remains in the
supernatant and can be recovered after centrifugation, as it is not affected by the neutralization
step.

5. Limitation: One of the limitations of the alkaline lysis method is the susceptibility of the plasmid
DNA to be contaminated with fragmented chromosomal DNA. Despite the selective denaturation
and precipitation of chromosomal DNA, there is a risk that small fragments of chromosomal DNA
may remain in the supernatant along with the plasmid DNA. This contamination can be a concern,
especially for downstream applications requiring pure plasmid DNA, such as cloning or transfection
experiments.

In summary, the alkaline lysis method is a widely used technique for extracting plasmid DNA from
bacterial cells. However, a limitation of this method is the potential for contamination of the purified
plasmid DNA with fragmented chromosomal DNA, which may require additional purification steps to
obtain a highly pure plasmid DNA sample.
Silica matrices

1. Principle of Silica Matrices DNA Extraction: The technique of DNA extraction using silica matrices is
based on the principle of selective binding of negatively charged DNA molecules to the silica surface,
which is coated with positively charged ions. This affinity between silicates and DNA was first
described by Vogelstein and Gillespie in 1979. The binding of DNA to the silica matrix allows for the
removal of cellular contaminants, enabling the extraction of purified DNA.

2. Extraction Process: After the DNA binds tightly to the silica matrix, the cellular contaminants can
be washed away, leaving the DNA attached to the silica particles. The extracted DNA is then eluted
from the silica particles using a suitable elution buffer, such as distilled water or a buffer like Tris-
EDTA.

3. Advantages of the Method: The silica matrices DNA extraction method offers several advantages,
including simplicity, speed, cost-efficiency, and the production of high-quality DNA. Additionally, this
technique can be easily adapted for automation, making it suitable for high-throughput DNA
extraction processes. Commercially available kits, such as the Thermo Fisher Purelink Genomic DNA
extraction kit and QIAGEN DNeasy Blood, utilize silica matrices for DNA extraction.

4. Limitations and Modifications: A major limitation of the silica matrices method is that the silica
particles cannot be reused, unlike the resins used in the anion exchange method. This is due to the
reduced binding capacity of silica matrices and the tendency for DNA particles to remain attached to
the silica even after elution. However, newer methods, such as maxXbond, have been developed to
allow for multiple uses of the silica matrices, addressing this limitation.

5. Modified Protocols: Various modified protocols for DNA extraction using silica matrices have been
described in the literature, including the use of hydrated silica matrix, which may offer improved
efficiency or specificity for certain applications.

In summary, the DNA extraction method using silica matrices is a widely used technique due to its
simplicity, speed, cost-efficiency, and ability to produce high-quality DNA. While the method has
limitations related to the reuse of silica matrices, newer technologies are addressing this issue,
making it a versatile and effective approach for DNA extraction in research and clinical settings.

Salting out method

1. Principle of Salting-Out DNA Extraction: The salting-out method is a non-toxic DNA extraction
technique described by Miller, Dykes, and Polesky in 1988. It is based on the principle of using high
salt concentrations to precipitate proteins, allowing the DNA to be separated and recovered from
the sample.
2. Extraction Process: In the salting-out method, the DNA-containing sample is initially added to a
lysis buffer containing 0.4 M NaCl, 10 mM Tris–HCl pH 8.0, and 2 mM EDTA, along with SDS and
proteinase K. The mixture is then incubated at an elevated temperature (55–65°C) overnight to
facilitate cell lysis and protein digestion. Subsequently, a high concentration of saturated NaCl
(approximately 6M) is added to the mixture, leading to a decrease in protein solubility and causing
their precipitation. The mixture is then centrifuged to separate the precipitated proteins from the
DNA-containing supernatant, which can be further processed for DNA precipitation using ethanol.

3. Advantages of the Method: The salting-out method has been reported to yield high-quality DNA
comparable to that obtained using the phenol-chloroform method. It is considered superior to the
phenol-chloroform method in terms of being more time-efficient, cost-effective, and, importantly,
the reagents used are non-toxic. This makes the salting-out method particularly advantageous for
routine DNA extractions in research and clinical laboratories, where safety and cost considerations
are important.

4. Versatility: The salting-out method is versatile and can be used to extract DNA from various
sources, including blood, suspension cultures, and tissue homogenates. This broad applicability
makes it a widely used technique for DNA extraction from diverse biological samples.

5. Safety and Environmental Considerations: The use of non-toxic reagents in the salting-out method
not only ensures the safety of laboratory personnel but also contributes to environmental
sustainability by reducing the potential hazards associated with toxic chemicals.

In summary, the salting-out method is a valuable non-toxic DNA extraction technique that offers
high-quality DNA yields, cost-effectiveness, and safety advantages over traditional methods. Its
versatility and compatibility with various sample types make it a preferred choice for DNA extraction
in many research and clinical settings.

CTAB method

1. Principle of CTAB DNA Extraction: The CTAB (cetyltrimethylammonium bromide) extraction

method, introduced by Doyle et al. in 1990, is a technique used for DNA extraction from samples
containing high amounts of polysaccharides, such as plants and bacteria. The method is based on the
precipitation of DNA and acidic polysaccharides from the rest of the cellular components using a
solution of low ionic strength containing 2% CTAB at alkaline pH.

2. Extraction Process: In the CTAB method, DNA-containing samples are treated with the extraction
buffer containing CTAB at alkaline pH. The low ionic strength of the solution causes the precipitation
of DNA and acidic polysaccharides, separating them from other cellular components. Subsequently,
high salt concentrations are used to remove DNA from the acidic polysaccharides, which form a
precipitate with CTAB. The DNA is then purified using various organic solvents and alcohols,
including toxic agents such as phenol and chloroform.

3. Suitability for Specific Samples: The CTAB method is particularly useful for DNA extraction from
plants and bacteria that produce high amounts of polysaccharides. The ability of CTAB to effectively
precipitate DNA and polysaccharides from complex plant and bacterial samples makes this method
valuable for researchers working with these organisms.

4. Limitations: One of the major limitations of the CTAB method is the use of toxic reagents,
including phenol and chloroform, for DNA purification. These agents pose potential health and
safety risks to laboratory personnel and require careful handling and disposal. Additionally, the
protocol for CTAB extraction is time-consuming, which can be a drawback in situations where rapid
DNA extraction is required.

5. Considerations for Use: Despite its limitations, the CTAB method remains a valuable technique for
DNA extraction from polysaccharide-rich samples. Researchers using this method should be mindful
of the potential hazards associated with toxic reagents and take appropriate safety precautions.
Additionally, the time-consuming nature of the protocol should be considered when planning
experiments and workflows.

In summary, the CTAB DNA extraction method is a valuable tool for researchers working with plant
and bacterial samples rich in polysaccharides. While it offers specific advantages for these sample
types, such as effective DNA precipitation, its use of toxic reagents and time-consuming protocol are
important considerations for researchers employing this technique.

Phenol chloroform methods

The phenol-chloroform DNA extraction method, introduced in 1998 by Barker et al., is a widely used
technique for isolating DNA from various sources, including blood, suspension cultures, and tissue
homogenates. The method involves several key steps to dissolve cell membranes, denature proteins,
and separate DNA from other cellular components. Here's a summary of the phenol-chloroform DNA
extraction method and its characteristics:

1. Cell Lysis: Cells are treated with a lysis buffer containing detergents such as sodium dodecyl
sulfate (SDS) to dissolve cell membranes and the nuclear envelope. The lysis buffer may also contain
Tris, EDTA, and NaCl.

2. Addition of Phenol-Chloroform-Isoamyl Alcohol (PCIA) Reagent: PCIA reagent is added in a specific

ratio to the sample. Phenol and chloroform efficiently denature proteins, while isoamyl alcohol
prevents emulsification and facilitates DNA precipitation.
3. Formation of Biphasic Emulsion: The contents are mixed to form a biphasic emulsion, which
separates into two phases upon centrifugation: an upper aqueous phase containing DNA and a
bottom organic phase containing proteins and other hydrophobic cellular components.

4. DNA Concentration: The DNA in the aqueous phase can be concentrated using ethanol
precipitation, followed by washing with cold ethanol to remove excess salt.

5. Advantages: The phenol-chloroform method is considered the gold standard for DNA extraction,
providing high yields and relatively low costs. It can be used for a wide range of sample types and
has been commercially adapted into kits such as Thermo Fisher Easy-DNA®.

6. Limitations: One major limitation of the phenol-chloroform method is the use of toxic reagents,
specifically phenol and chloroform, which require the use of fume hoods and careful handling.
Additionally, while the method yields highly pure DNA, the purity may be lower compared to DNA
obtained using column-based methods.

In summary, the phenol-chloroform DNA extraction method is a robust and cost-effective technique
that provides high DNA yields from diverse sample types. However, the use of toxic reagents and the
need for careful handling are important considerations. Despite its limitations, the method remains
widely used and has been adapted into commercial kits for DNA extraction.

Sds protenase k method

1. Proteinase K: Proteinase K is a serine protease that effectively degrades proteins and is commonly
used in DNA extraction protocols. It was first discovered in the fungus Engyodontium album in 1974.
Proteinase K is added to the DNA extraction solution at a concentration of 10-20 mg/mL to digest
proteins and facilitate the release of DNA.

2. Sodium Dodecyl Sulfate (SDS): SDS is an anionic detergent that is added to the DNA extraction
solution to dissolve the cell membrane and nuclear envelope. Additionally, SDS denatures and
unfolds proteins, exposing them to the protease activity of proteinase K. This step is crucial for
breaking down cellular structures and releasing DNA for subsequent extraction.

3. Incubation: After the addition of proteinase K and SDS, the solution is incubated for 1-18 hours at
a temperature range of 50-60°C. During this incubation period, proteinase K digests cellular proteins,
while SDS disrupts the cell membrane and denatures proteins, allowing for efficient DNA release.

4. DNA Extraction: Following the incubation, the solution can be used for DNA extraction using
methods such as phenol-chloroform or salting-out. These methods allow for the separation of DNA
from other cellular components, resulting in purified DNA that can be further processed for
downstream applications.

In summary, the combination of proteinase K and SDS is a critical step in DNA extraction protocols.
Proteinase K digests proteins, while SDS dissolves the cell membrane and denatures proteins,
ultimately facilitating the release of DNA. This process is essential for obtaining high-quality DNA for
various molecular biology applications.
Silica based method

The silica column-based DNA extraction method is a widely used technique for isolating DNA from
various sources, offering several advantages over conventional organic solvent-based methods.
Here's an explanation of the method and its key points:

1. Lysis and Protein Digestion: The sample is initially treated with lysis buffer containing EDTA, Tris,
SDS, and Proteinase K. The lysis buffer helps break down cell membranes and release cellular
components, while SDS and Proteinase K work to denature proteins and digest them, respectively.

2. Addition of Silica Gel: After the lysis and protein digestion, the sample is added to a tube
containing silica gel. Silica gel has a high affinity for DNA and allows for efficient binding of DNA to
the gel, while proteins and other cellular components are retained beneath the silica column.

3. Phenol-Chloroform Extraction: A mixture of phenol-chloroform is added to the sample at a 1:1

ratio. This mixture is then vigorously shaken and centrifuged to separate the aqueous phase
containing DNA from the proteins and organic phase. The DNA-containing aqueous layer remains
above the layer of gel polymer due to the trapping of proteins and organic components beneath the
silica column.

4. DNA Recovery: The DNA-containing aqueous layer is carefully transferred to a new tube, typically
by decanting or pipetting, and then dissolved in a buffer such as TE buffer. The use of silica gel in this
method helps to increase the purity of the extracted DNA by preventing contamination and physical
contact with toxic reagents.

5. Advantages of Silica Column-Based Method: The use of silica gel in DNA extraction helps to
increase the purity of the extracted DNA by eliminating the interface where contamination can occur
in conventional methods. Additionally, the method reduces the need for handling toxic organic
solvents, thereby enhancing safety. Furthermore, the DNA yield using the silica column-based
method has been reported to be 40% higher compared to traditional organic solvent-based DNA
extraction methods.

In summary, the silica column-based DNA extraction method offers improved purity, safety, and
higher DNA yield compared to conventional organic solvent-based methods. The use of silica gel
efficiently separates DNA from other cellular components, making it a valuable technique for various
molecular biology applications.
Magnetic bead based method

The use of magnetic beads for DNA purification and isolation offers several advantages over
conventional methods, as well as some considerations to keep in mind. Here's an explanation of the
method and its key points:

1. Principle and Composition: Magnetic nanoparticles coated with a DNA binding antibody or
polymer with specific affinity to DNA are used to bind DNA to their surface. These beads are
typically composed of magnetite or maghemite at the core, with surface substances such as
silica, sulphate, and hydroxyl groups. The specific affinity of the coating material for DNA
allows for efficient binding and isolation of DNA from the sample.
The attachment of hydroxyl groups to the surface of magnetic beads in DNA extraction procedures serves to provide a platform for covalent attachment of
molecules such as DNA capture probes or primers. This functionalization of the beads allows for the selective binding of DNA or RNA molecules, enabling their
isolation from complex mixtures.

While it's true that hydroxyl groups are negatively charged, the interaction between the hydroxyl groups on the bead surface and DNA is not primarily driven
by electrostatic forces. Instead, it involves a combination of hydrogen bonding, hydrophobic interactions, and van der Waals forces.

Hydroxyl groups can form hydrogen bonds with the phosphate backbone of DNA, contributing to the binding of DNA to the bead surface. Additionally,
hydrophobic interactions between the nonpolar regions of DNA bases and the hydrophobic portions of the bead surface can also play a role in the binding
process. Van der Waals forces, which are attractive forces between molecules due to temporary dipoles, also contribute to the interaction between the
hydroxyl-functionalized bead surface and DNA molecules.

Overall, the attachment of hydroxyl groups to the surface of magnetic beads provides functional groups that enable the selective binding of DNA through a
combination of different intermolecular forces, rather than relying solely on electrostatic interactions.

2. Separation and Purification: After DNA binding, the DNA-bound magnetic beads are separated
from the cell lysate by applying a magnetic field at the bottom of the tube using an external magnet.
The beads aggregate at the bottom of the tube, allowing the supernatant to be rinsed away,
effectively purifying the DNA.

3. Elution and Efficiency: The magnetic pellet containing the DNA-bound beads can be eluted via the
ethanol precipitation method. Subsequently, the pellet can be incubated at 65°C to elute the
magnetic particles from the DNA. The DNA yield obtained by this method is comparable to that
obtained in other conventional methods, and the protocol has been shown to be much faster,
typically taking less than 15 minutes to complete, which is significantly quicker than traditional
methods that can take several hours.

4. Automation and Equipment: The magnetic bead-based method is ideal for automation and
requires minimal equipment, as it does not depend on centrifugation. Additionally, it does not
involve the use of shear forces that could potentially damage the integrity of nucleic acids, as seen in
centrifugation-based methods.

5. Cost Considerations: One drawback of this method is that it may not be as cost-effective as some
other conventional methods. The initial investment in the magnetic beads and associated reagents
can be higher, which may be a consideration for some laboratories.
In summary, the use of magnetic beads for DNA purification and isolation offers advantages such as
rapid processing, minimal equipment requirements, and preservation of DNA integrity. However,
cost considerations should be taken into account when choosing this method. Overall, it is a valuable
technique for efficient and high-quality DNA isolation.

Cellulose paper based method

The use of cellulose-based paper for DNA extraction, such as the Whatman™ FTA™ Cards, offers
certain advantages, but it also presents challenges in downstream processing for pure and
concentrated DNA recovery. Here's an explanation of the method and its key points:

1. DNA Extraction Process: Cellulose-based paper, such as FTA™ Cards, is impregnated with buffers,
detergents, and chelating agents that facilitate the extraction of DNA. The sample, typically a
biological specimen containing DNA, is applied to the paper, and about 1-2 mm of the sample area is
removed using a sterilized micropunch. The punch is then washed with detergent and rinsed before
further downstream processing.

2. Advantages: DNA extraction using cellulose-based paper is fast, highly convenient, and does not
require profound laboratory expertise. Additionally, samples collected on FTA™ Cards can be stored
without refrigeration, making them suitable for field applications and remote settings.

3. Downstream Processing Challenges: While the initial DNA extraction process is convenient,
downstream processing to recover pure and concentrated DNA from the cellulose-based paper can
be challenging. The DNA recovery process from these cards may be less straightforward compared to
other extraction methods, and it may require additional steps to achieve high purity and
concentration of DNA.

4. Sample Storage: Although FTA™ Cards enable easy storage of samples without refrigeration, the
recovery of DNA from these cards for downstream applications, such as PCR or sequencing, may
require specific protocols and optimization to achieve the desired purity and concentration.

5. Considerations for Downstream Applications: Laboratories using cellulose-based paper for DNA
extraction should consider the downstream applications and requirements for pure and
concentrated DNA. Additional purification steps or specialized protocols may be necessary to ensure
the DNA is suitable for specific downstream applications.

In summary, cellulose-based paper offers a convenient and easy method for DNA extraction and
sample storage, particularly in field and remote settings. However, laboratories should be aware of
the challenges associated with downstream processing to recover pure and concentrated DNA, and
be prepared to address these challenges through appropriate methods and protocols.
Cellulose-based extraction of DNA utilizes the unique properties of cellulose, a naturally occurring polysaccharide, to
capture and purify DNA from biological samples. The process typically involves the use of cellulose columns or membranes,
and it can be employed in various DNA extraction protocols. Here's an overview of how cellulose-based DNA extraction
works:

1. Binding of DNA: Cellulose possesses a high affinity for nucleic acids, particularly DNA, due to its ability to form hydrogen
bonds with the phosphate backbone of DNA molecules. In the extraction process, the biological sample (e.g., tissue, blood,
or cultured cells) is lysed to release the DNA. The lysate is then applied to the cellulose matrix, where the DNA binds to the
cellulose through hydrogen bonding and other interactions.

2. Washing: After the DNA has been captured by the cellulose matrix, the column or membrane is washed to remove
contaminants such as proteins, lipids, and other cellular debris. This step helps to purify the DNA and remove substances
that could interfere with downstream applications.

3. Elution of DNA: The purified DNA is then eluted from the cellulose matrix using a suitable elution buffer. The elution
buffer disrupts the interactions between the DNA and cellulose, allowing the DNA to be released into the eluate. The
eluted DNA is now in a form suitable for use in various molecular biology applications, such as PCR, sequencing, or
restriction enzyme digestion.

Chelax-100 based extraction

The DNA extraction method using Chelex-100 offers certain advantages, such as reduced risk for
contamination and mis-pipetting, but it also has limitations, particularly in terms of purification and
stability of the isolated DNA. Here's an explanation of the method's advantages and limitations:

1. DNA Extraction Process: The Chelex-100 extraction method involves using a 5% Chelex solution
and proteinase K to degrade DNases. The tissue sample is then boiled in the Chelex solution to lyse
membranes, denature proteins, and release DNA. The Chelex resin chelates metal ions that act as
cofactors for DNases, stabilizing the DNA preparation. The resulting single-stranded DNA can be
concentrated from the supernatant after centrifugation.
2. Advantages: The method offers reduced risk for contamination and mis-pipetting, as the
procedure involves the use of a single test tube and few manipulating steps. This reduces the
potential for errors and contamination during the extraction process, making it a convenient method
for certain applications.

3. Limitations in Purification: One of the limitations of the Chelex-100 extraction method is the lack
of purification. While it efficiently stabilizes the DNA and prevents it from being digested by residual
DNases, it does not effectively remove PCR inhibitors or other contaminants that may be present in
the sample. This can be a drawback for downstream applications that require pure DNA, such as PCR
or sequencing.

4. DNA Stability: The isolated DNA obtained using the Chelex-100 method may be unstable,
particularly for certain downstream applications. The lack of purification and the presence of
residual components from the tissue sample and extraction process can affect the stability and
suitability of the DNA for certain analyses. For example, the DNA may not be suitable for restriction
fragment length polymorphism (RFLP) analysis, which requires highly pure and intact DNA.

5. Considerations for Downstream Applications: Laboratories using the Chelex-100 method should
consider the specific requirements of downstream applications. If high-purity DNA is needed for
applications like RFLP analysis, additional purification steps may be necessary. Additionally, the
potential presence of PCR inhibitors should be considered for applications like PCR, as the isolated
DNA may not be suitable for amplification without further purification.

In summary, while the Chelex-100 extraction method offers advantages in terms of reduced
contamination risk and simplicity, it has limitations related to purification and DNA stability.
Laboratories should carefully assess the specific needs of downstream applications and consider
additional purification steps to ensure the isolated DNA is suitable for the desired analyses.

Filter paper based extraction

The filter paper-based DNA extraction method described by Shi and Panthee in 2017 offers a cost-
effective alternative to commercial kits for DNA extraction from plant sources. Here's an explanation
of the method and its advantages:

1. DNA Extraction Process: The filter paper-based DNA extraction method involves using a spin plate
composed of a 96-well plate with a hole drilled into the bottom of each well. Each well contains a
disc of Whatman filter paper. Samples are treated with lysis buffers and filtered with centrifugation,
allowing the DNA to bind to the filter paper. The filter paper is then washed and the DNA is eluted
for downstream applications.

2. Advantages: The filter paper-based method offers a cost-effective alternative to commercial kits
for DNA extraction, as it replaces expensive glass fiber filters with inexpensive filter paper. This
greatly reduces the costs of the method, making it accessible to laboratories with limited budgets.
Additionally, the method is simple and efficient, allowing for high-throughput extraction of DNA
from plant sources.

3. Limitations: One potential limitation of the filter paper-based method is the potential for
variability in DNA yield and quality depending on the type of filter paper used and the specific lysis
buffers and protocols employed. Additionally, the method may not be suitable for certain
downstream applications that require highly pure and concentrated DNA.

4. Considerations for Downstream Applications: Laboratories using the filter paper-based method
should consider the specific requirements of downstream applications. If highly pure and
concentrated DNA is needed, additional purification steps may be necessary. Additionally, the
potential presence of PCR inhibitors should be considered for applications like PCR, as the isolated
DNA may not be suitable for amplification without further purification.

In summary, the filter paper-based DNA extraction method offers a cost-effective and efficient
alternative to commercial kits for DNA extraction from plant sources. Laboratories should carefully
assess the specific needs of downstream applications and consider additional purification steps to
ensure the isolated DNA is suitable for the desired analyses.