DNA extraction is a method used to purify DNA from a sample by separating it from other cellular components using physical and chemical techniques. It involves lysing cells and solubilizing the DNA using detergents and enzymes. Common extraction methods include organic extraction, non-organic extraction, and adsorption. DNA can then be extracted from various samples for purposes like genetic testing, forensic analysis, and body identification.
Basics of DNA isolation, What is chemistry behind it. Presently the laboratory of animal science department ,Göttingen university using this technique for dna isolation in pig blood sample.
Esta presentación la encontré en la web y entiendo que es muy útil para la práctica de Electroforesis de Gel Agarosa. Explica de forma básica de que consta esta tecnica molecular y los pasos a seguir.
The change in one nucleotide in a genome is known as single nucleotide polymorphism. There are assorted types of SNPs. SNPs can be detected by several analytical techniques i.e. DNA sequencing, microchip, HPLC and oligonucleotide ligation reaction.
Basics of DNA isolation, What is chemistry behind it. Presently the laboratory of animal science department ,Göttingen university using this technique for dna isolation in pig blood sample.
Esta presentación la encontré en la web y entiendo que es muy útil para la práctica de Electroforesis de Gel Agarosa. Explica de forma básica de que consta esta tecnica molecular y los pasos a seguir.
The change in one nucleotide in a genome is known as single nucleotide polymorphism. There are assorted types of SNPs. SNPs can be detected by several analytical techniques i.e. DNA sequencing, microchip, HPLC and oligonucleotide ligation reaction.
Enzymes that cut DNA at or near specific recognition nucleotide sequences known as restriction sites.
Especial class of enzymes that cleave (cut) DNA at a specific unique internal location along its length.
Often called restriction endonucleases (Because they cut within the molecule).
Discovered in the late 1970s by Werner Arber, Hamilton Smith, and Daniel Nathans.
Essential tools for recombinant DNA technology.
Naturally produced by bacteria that use them as a defense mechanism against viral infection.
Chop up the viral nucleic acids and protect a bacterial cell by hydrolyzing phage DNA.
DNA Fingerprinting of plants . History,procedure of DNA fingerprinting, PCR and NON PCR technique like RAPD,SSR,RELPs, application of DNA fingerprinting, advantage and disadvantage of DNA fingerprinting.
this section helps students how to isolate DNA from various sources. specially life life science fields such as biotechnology, biology, and medical laboratory
This lectureis about DNA extraction from whole Blood presented by Tuba nafees she is msc graduate in Biotechnology from University of Karachi, Sindh Pakistan.
lecture video is also there in youtube link:
https://www.youtube.com/watch?v=cGr__SuqYgY&t=409s
A class of DNA sequencing techniques currently in active development is third-generation sequencing, commonly referred to as long-read sequencing. In comparison to second generation sequencing, also referred to as next generation sequencing, third generation sequencing technologies have the capacity to create noticeably longer reads.
Techniques of DNA Extraction, Purification and QuantificationBHUMI GAMETI
Introduction
The overall process…
Uses of isolated genomic DNA
Extraction of DNA from plant material
Components of DNA extraction solutions
Cell Lysis or Cell disruption :
Purification of DNA
CTAB Method
Phenol–chloroform extraction
PROTEINASE K
Salting out
Silica adsorption method
Magnetic beads
FTA Paper
Nucleic acid quantification
Agarose Gel Electrophoresis
UV spectroscopy
DNA quantification using NanoDrop
Lab 23 DNA Extraction and PurificationIsolation and purific.docxDIPESH30
Lab 2/3: DNA Extraction and Purification
Isolation and purification of nucleic acids is the most fundamental procedure in molecular biology. There are three basic steps involved:
1. Lyse (break open) the cells (and nuclei in eukaryotes) to release the DNA
2. Remove contaminants (proteins, lipids, carbohydrates, salts)
3. Preserve the integrity of the DNA (prevent degradation and shearing)
Step 1 can be accomplished in a number of ways, such as mechanical disruption (grinding, mincing), protein denaturation (detergents), and protein degradation (via proteases). These can be used singly or in combination depending on the type of biological sample you are starting with. Grinding the samples provides more surface area for the denaturants/proteases to interact with the cellular proteins, thus speeding up the denaturation process. We used liquid nitrogen (N2) and protein degradation (Proteinase K) in lab 2. Various salts are included in a cell lysis solution to stabilize the DNA by providing positive ions which insert between the negatively charged phosphates in the DNA backbone (creating a “salt bridge”). Buffers (such as Tris) also help to preserve DNA integrity by maintaining a neutral pH.
Once the cells have been lysed, contaminating proteins, lipids, etc. must be separated from the DNA. A widely used and efficient way to remove proteins from nucleic acids solutions is to extract with a 1:1 mixture of phenol and chloroform (CHCl3). Phenol and CHCl3 are both hydrophobic organic solvents that unfold proteins. When mixed with an aqueous DNA/protein solution and then centrifuged, the denatured proteins are selectively partitioned into the denser organic phase, while the DNA (plus RNA and salt) remains in the aqueous phase. This procedure takes advantage of the fact that deproteinization is more efficient when two different organic solvents are used instead of one. Additionally, chloroform removes any lingering traces of phenol from the nucleic acid preparation (which would interfere with later applications). Since the aqueous phase contains RNA and salt in addition to the DNA, phenol:CHCl3 extraction is followed by ethanol (EtOH) precipitation. DNA (a polar molecule) is soluble in water (also polar) because the water molecules intercalate into the phosphate backbone of the DNA and thus maintain it in a soluble state, but DNA is insoluble in 95% EtOH (nonpolar). Water molecules have a higher affinity for the EtOH than the DNA, so when you add EtOH and salt [10 M ammonium acetate (NH4Ac); pH 5.2], Na+ ions replace water in the DNA backbone, essentially removing the water molecules, and the DNA is forced out of solution (precipitates). After precipitating with 95% EtOH, the DNA is “washed” in 70% EtOH to remove the salt. Since 70% EtOH contains 30% water, the salt, having a greater affinity for the water than the DNA, remains in the EtOH, and the DNA is forced out.
The final step in the purification process is to preserve the DNA in a sta ...
Enzymes that cut DNA at or near specific recognition nucleotide sequences known as restriction sites.
Especial class of enzymes that cleave (cut) DNA at a specific unique internal location along its length.
Often called restriction endonucleases (Because they cut within the molecule).
Discovered in the late 1970s by Werner Arber, Hamilton Smith, and Daniel Nathans.
Essential tools for recombinant DNA technology.
Naturally produced by bacteria that use them as a defense mechanism against viral infection.
Chop up the viral nucleic acids and protect a bacterial cell by hydrolyzing phage DNA.
DNA Fingerprinting of plants . History,procedure of DNA fingerprinting, PCR and NON PCR technique like RAPD,SSR,RELPs, application of DNA fingerprinting, advantage and disadvantage of DNA fingerprinting.
this section helps students how to isolate DNA from various sources. specially life life science fields such as biotechnology, biology, and medical laboratory
This lectureis about DNA extraction from whole Blood presented by Tuba nafees she is msc graduate in Biotechnology from University of Karachi, Sindh Pakistan.
lecture video is also there in youtube link:
https://www.youtube.com/watch?v=cGr__SuqYgY&t=409s
A class of DNA sequencing techniques currently in active development is third-generation sequencing, commonly referred to as long-read sequencing. In comparison to second generation sequencing, also referred to as next generation sequencing, third generation sequencing technologies have the capacity to create noticeably longer reads.
Techniques of DNA Extraction, Purification and QuantificationBHUMI GAMETI
Introduction
The overall process…
Uses of isolated genomic DNA
Extraction of DNA from plant material
Components of DNA extraction solutions
Cell Lysis or Cell disruption :
Purification of DNA
CTAB Method
Phenol–chloroform extraction
PROTEINASE K
Salting out
Silica adsorption method
Magnetic beads
FTA Paper
Nucleic acid quantification
Agarose Gel Electrophoresis
UV spectroscopy
DNA quantification using NanoDrop
Lab 23 DNA Extraction and PurificationIsolation and purific.docxDIPESH30
Lab 2/3: DNA Extraction and Purification
Isolation and purification of nucleic acids is the most fundamental procedure in molecular biology. There are three basic steps involved:
1. Lyse (break open) the cells (and nuclei in eukaryotes) to release the DNA
2. Remove contaminants (proteins, lipids, carbohydrates, salts)
3. Preserve the integrity of the DNA (prevent degradation and shearing)
Step 1 can be accomplished in a number of ways, such as mechanical disruption (grinding, mincing), protein denaturation (detergents), and protein degradation (via proteases). These can be used singly or in combination depending on the type of biological sample you are starting with. Grinding the samples provides more surface area for the denaturants/proteases to interact with the cellular proteins, thus speeding up the denaturation process. We used liquid nitrogen (N2) and protein degradation (Proteinase K) in lab 2. Various salts are included in a cell lysis solution to stabilize the DNA by providing positive ions which insert between the negatively charged phosphates in the DNA backbone (creating a “salt bridge”). Buffers (such as Tris) also help to preserve DNA integrity by maintaining a neutral pH.
Once the cells have been lysed, contaminating proteins, lipids, etc. must be separated from the DNA. A widely used and efficient way to remove proteins from nucleic acids solutions is to extract with a 1:1 mixture of phenol and chloroform (CHCl3). Phenol and CHCl3 are both hydrophobic organic solvents that unfold proteins. When mixed with an aqueous DNA/protein solution and then centrifuged, the denatured proteins are selectively partitioned into the denser organic phase, while the DNA (plus RNA and salt) remains in the aqueous phase. This procedure takes advantage of the fact that deproteinization is more efficient when two different organic solvents are used instead of one. Additionally, chloroform removes any lingering traces of phenol from the nucleic acid preparation (which would interfere with later applications). Since the aqueous phase contains RNA and salt in addition to the DNA, phenol:CHCl3 extraction is followed by ethanol (EtOH) precipitation. DNA (a polar molecule) is soluble in water (also polar) because the water molecules intercalate into the phosphate backbone of the DNA and thus maintain it in a soluble state, but DNA is insoluble in 95% EtOH (nonpolar). Water molecules have a higher affinity for the EtOH than the DNA, so when you add EtOH and salt [10 M ammonium acetate (NH4Ac); pH 5.2], Na+ ions replace water in the DNA backbone, essentially removing the water molecules, and the DNA is forced out of solution (precipitates). After precipitating with 95% EtOH, the DNA is “washed” in 70% EtOH to remove the salt. Since 70% EtOH contains 30% water, the salt, having a greater affinity for the water than the DNA, remains in the EtOH, and the DNA is forced out.
The final step in the purification process is to preserve the DNA in a sta ...
electrophoresis: types, advantages, disadvantages and applications.Cherry
Electrophoresis is a general term that describes the migration and separation of charged particles under the influence of an electric field.
The particles maybe simple ions, complex macromolecules and colloids or particulate matter- either living cells such as bacteria or inert material such as oil emulsion, droplet etc.
The pores present in the gel work like a sieve, allowing the smaller molecules to pass through more quickly and easily than the larger molecules.
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Health Education on prevention of hypertensionRadhika kulvi
Hypertension is a chronic condition of concern due to its role in the causation of coronary heart diseases. Hypertension is a worldwide epidemic and important risk factor for coronary artery disease, stroke and renal diseases. Blood pressure is the force exerted by the blood against the walls of the blood vessels and is sufficient to maintain tissue perfusion during activity and rest. Hypertension is sustained elevation of BP. In adults, HTN exists when systolic blood pressure is equal to or greater than 140mmHg or diastolic BP is equal to or greater than 90mmHg. The
Deep Leg Vein Thrombosis (DVT): Meaning, Causes, Symptoms, Treatment, and Mor...The Lifesciences Magazine
Deep Leg Vein Thrombosis occurs when a blood clot forms in one or more of the deep veins in the legs. These clots can impede blood flow, leading to severe complications.
How many patients does case series should have In comparison to case reports.pdfpubrica101
Pubrica’s team of researchers and writers create scientific and medical research articles, which may be important resources for authors and practitioners. Pubrica medical writers assist you in creating and revising the introduction by alerting the reader to gaps in the chosen study subject. Our professionals understand the order in which the hypothesis topic is followed by the broad subject, the issue, and the backdrop.
https://pubrica.com/academy/case-study-or-series/how-many-patients-does-case-series-should-have-in-comparison-to-case-reports/
CHAPTER 1 SEMESTER V - ROLE OF PEADIATRIC NURSE.pdfSachin Sharma
Pediatric nurses play a vital role in the health and well-being of children. Their responsibilities are wide-ranging, and their objectives can be categorized into several key areas:
1. Direct Patient Care:
Objective: Provide comprehensive and compassionate care to infants, children, and adolescents in various healthcare settings (hospitals, clinics, etc.).
This includes tasks like:
Monitoring vital signs and physical condition.
Administering medications and treatments.
Performing procedures as directed by doctors.
Assisting with daily living activities (bathing, feeding).
Providing emotional support and pain management.
2. Health Promotion and Education:
Objective: Promote healthy behaviors and educate children, families, and communities about preventive healthcare.
This includes tasks like:
Administering vaccinations.
Providing education on nutrition, hygiene, and development.
Offering breastfeeding and childbirth support.
Counseling families on safety and injury prevention.
3. Collaboration and Advocacy:
Objective: Collaborate effectively with doctors, social workers, therapists, and other healthcare professionals to ensure coordinated care for children.
Objective: Advocate for the rights and best interests of their patients, especially when children cannot speak for themselves.
This includes tasks like:
Communicating effectively with healthcare teams.
Identifying and addressing potential risks to child welfare.
Educating families about their child's condition and treatment options.
4. Professional Development and Research:
Objective: Stay up-to-date on the latest advancements in pediatric healthcare through continuing education and research.
Objective: Contribute to improving the quality of care for children by participating in research initiatives.
This includes tasks like:
Attending workshops and conferences on pediatric nursing.
Participating in clinical trials related to child health.
Implementing evidence-based practices into their daily routines.
By fulfilling these objectives, pediatric nurses play a crucial role in ensuring the optimal health and well-being of children throughout all stages of their development.
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India Clinical Trials Market: Industry Size and Growth Trends [2030] Analyzed...Kumar Satyam
According to TechSci Research report, "India Clinical Trials Market- By Region, Competition, Forecast & Opportunities, 2030F," the India Clinical Trials Market was valued at USD 2.05 billion in 2024 and is projected to grow at a compound annual growth rate (CAGR) of 8.64% through 2030. The market is driven by a variety of factors, making India an attractive destination for pharmaceutical companies and researchers. India's vast and diverse patient population, cost-effective operational environment, and a large pool of skilled medical professionals contribute significantly to the market's growth. Additionally, increasing government support in streamlining regulations and the growing prevalence of lifestyle diseases further propel the clinical trials market.
Growing Prevalence of Lifestyle Diseases
The rising incidence of lifestyle diseases such as diabetes, cardiovascular diseases, and cancer is a major trend driving the clinical trials market in India. These conditions necessitate the development and testing of new treatment methods, creating a robust demand for clinical trials. The increasing burden of these diseases highlights the need for innovative therapies and underscores the importance of India as a key player in global clinical research.
2. Genetic
testing
Body
identification
Analysis of
a forensic
evidence
DNA Extraction
• DNA extraction is a method use to purify DNA by using physical and/or chemical methods from a sample
separating DNA from cell membranes, proteins, and other cellular components.
• It involves lysing the cells and solubilizing DNA, which is followed by chemical or enzymatic methods to
remove macromolecules, lipids, RNA, or proteins.
DNA extraction techniques include organic extraction (phenol–chloroform method), nonorganic
method (salting out and proteinase K treatment), and adsorption method (silica–gel membrane).
3. The basic criteria that any method of DNA isolation from any sample
type should meet include:
(1) efficient extraction of DNA from the sample
(2) production of a sufficient amount of DNA for use in downstream
processes
(3) successful removal of contaminants
(4) isolation of high quality and high purity DNA.
Samples used for DNA extraction can be taken from human tissues,
blood, hair, rodent tissues, leaf tissue, bacteria, yeast, fungi, insect, stool, body
fluids, spores, soil, clinical samples (e.g., biopsy samples, fine needle aspirates),
forensic samples (e.g., dried blood spots, buccal swabs), and fingerprints.
INTRO
4. Collection of sample
Cell lysis
Precipitation of DNA
Removal of the DNA
FLOW CHART OF THE DNA EXTRACTION
Source: Retrieved November 22, 2022 from
https://learn.genetics.utah.edu/content/labs/extraction
LYSIS
SOLUTION
ISOPROPYL
SALT
SOLUTION
5. This composes of two chemicals:
a. Detergent - disrupts the cell membrane and nuclear
envelope.
b. Proteinase K - Hydrolyzes histones apart freeing DNA. It
inactivate DNases and RNases that would otherwise degrade
a desired DNA or RNA sample.
LYSIS SOLUTION
DNA’s sugar phosphate backbone is charged. By adding salt,
it helps neutralize the DNA charge and make the molecule
less hydrophilic, meaning it becomes less soluble in water.
The salt also helps to remove proteins that are bound to the
DNA and to keep the proteins dissolved in the water.
SALT SOLUTION
Figure 1:
Figure 2:
6. It performs three (3) different functions in DNA extraction, which are precipitation
washing and storing.
It is because DNA is not soluble in alcohol, it allows the sodium ions to interact
with the DNA molecules more easily. Wherein, the colder the alcohol, the less soluble DNA
will be in it.
ISOPROPYL SOLUTION
Polar and nonpolar molecules dissolve in polar and
nonpolar solutions, respectively. Solute and solvents dissolve
properly only if they have a similar structure. DNA is a polar
molecule and soluble in water as water is also partially polar.
But water can destroy everything, the positive
charge of water will try to dissolve and stabilize it. So what
happens is that in the addition of alcohol, the net negative
charge of alcohol makes the positive charge of water busy.
This disallows water to interact with the DNA,
meaning, that alcohol prevents the Na and PO3– complex by
neutralizing other positive charges. With the addition of more
and more alcohol it neutralizes as many positive charges as
possible from the solution and precipitates DNA in a visible
form.
7. What is the purpose of the warm water bath in
DNA extraction?
The warm water bath helps the DNA to be freed from the cells. The warm water will melt the membranes and then afterwards
eethe soap can be added to dissolve the lipid layers.
Why does the DNA sink to the bottom of the tube
after it is being centrifuged?
The centrifuge aids in separating the DNA and its impurities based on their difference in molecular weight. Wherein, the
molecular weight of DNA is lighter than the other cell material, like proteins and cell walls. So, by spinning the sample with
centrifuge, the material from the DNA can be separated thus a cleaner DNA sample.
What does the centrifuge do in the process? Why
does it needs to be balanced?
The water is “pushed” against the bottom of the bucket by what is colloquially known as “centrifugal force.”
Though not a real force, but a reaction to the centripetal force, centrifugal force pushes against the bottom of the bucket.
This causes the particles in the solution to clump at the bottom of the tube. That solid clump is called a pellet, and the
solution above it is called the “supernatant”.
8. 1. The molecules will travel through the gel in different directions or at different speeds based on what?
2. This is known as the filter that sorts the DNA strands which usually looks like a sponge that is made of a Jell – O.
3. What is the chemical name of Ethidium bromide?
4. Which DNA strands will move the farthest? that move the nearest?
5. This is the isolation of any sub – cell structures.
6. What are the three (3) processes involved in cell fractionation?
7. How long usually for the DNA to run in a gel electrophoresis?
8. What do the presence of ‘bubbles’ mean?
9. Based on your simulation at the website in ‘genetics Utah’, the period of how long should the gel electrophoresis runs
depends on what factors?
10.What is the purpose of the salt water solution?
11.What is the difference between fractionation and centrifugation?
12. Ethidium bromide is used as a what?
13. What is the relationship of the length of molecules and their speeds?
14. Gel electrophoresis separates DNA fragments based on their size and charge. In the process, the molecules or the DNA
strands is pushed through a gel that contains the small pores. What pushes the DNA strands into the small pores of the
gel?
GEL ELECTROPHORESIS
9. It is a technique used to separate DNA
fragments based on their sized and charge.
A filter that sorts the DNA strands and is like
a sponge made of a Jell – O with many small
holes in it.
GEL ELECTROPHORESIS
GEL
Electrophoresis
Refers to a technique that uses electrical
current to separate DNA, RNA or proteins
based on their physical properties such as
size and charge.
11. WHAT IS USED?
a. Agarose gel
A component of agar. It forms a 3D gel matrix of helical agarose
molecules in supercoiled bundles held by hydrogen bonds, with
channels and pores through which molecules are able to pass.
When heated, these hydrogen bonds break, turning the agarose
to liquid and allowing it to be poured into a mold before it resets.
12. • Percentage of agarose included in a gel impacts the pore sizes and thus the size of
molecules that may pass through and speed at which they do so. The higher the
percentage of agarose, the smaller the pore size, thus the smaller the molecules able to
pass and the slower the migration.
• 0.7-1% agarose gel is typically used for day-to-day DNA separations, offering good, clear
differentiation of fragments in the range of 0.2-10 kb. Larger fragments may be resolved
using lower percentage gels, but they become very fragile and hard to handle, while
higher percentage gels will give better resolution of small fragments but are brittle and
may set unevenly.
13. b. Ethidium bromide
This is an intercalating dye and is incorporated in the gel during setting. This binds the
DNA and fluoresces under UV light, allowing the DNA fragments to be visualized. The
more DNA present, the brighter the band.
Key steps in Gel
Electrophoresis:
• Determine the required gel percentage
• Pour a gel
• Mix samples/ ladders with loading dye
• Load the gel
• Run the gel
• Visualization
14. HOW TO READ GEL
ELECTROPHORESIS
Base pairs are two complementary DNA
nucleotide bases that pair together to form a
“rung of the DNA ladder.”
In humans it ranges in size from about 50
million to 300 million base pairs. Although in 23
chromosomes, it only consists of approximately
3 billion base pairs of DNA.
15. Agarose gel (2%) analysis of PCR-amplified products
from DNA extracted from a bronchoalveolar lavage
(BAL) diagnostic specimen of a patient with pulmonary
symptoms. Credit: The Centers for Disease Control and
Prevention.
16. In the well, DNA is a negatively charged molecule due to the phosphate
groups that constitute the backbone of DNA, which is due to the presence
of bonds created between the phosphorus and oxygen atoms. Therefore,
by placing the DNA at the negative end of the gel matrix, when the current
is turned on the DNA will migrate down the gel towards the positive side
because the opposite charges attract.
17. IMPORTANCE OF GEL ELECTROPHORESIS
• Visualization of sample DNA
a. Whether DNA is present in a sample.
b. The sizes of DNA fragments present.
c. How clean the sample is.
• Separation of DNA fragments for purification
• Separation of DNA fragments for Southern blotting
• Electrophoretic mobility shift assays (EMSAs)