Agarose gel electrophoresis is a method used to separate DNA fragments by size using an electrical current. DNA carries a negative charge and will migrate towards the positive electrode through an agarose gel matrix. Shorter DNA fragments travel farther and faster than longer fragments. Agarose gel electrophoresis is used to analyze restriction enzyme digestion products and determine DNA fragment sizes between 500-30,000 base pairs. It has applications in forensic analysis, genetic disease diagnosis, and plant breeding.
An isotope is one of two or more atoms having the same atomic number but different mass numbers.
Unstable isotopes are called Radioisotopes.
uses of radioisotopes are many which are discussed in this slide.
An isotope is one of two or more atoms having the same atomic number but different mass numbers.
Unstable isotopes are called Radioisotopes.
uses of radioisotopes are many which are discussed in this slide.
Separation is brought about through molecular sieving technique, based on the molecular size of the substances. Gel material acts as a "molecular sieve”.
Gel is a colloid in a solid form (99% is water).
It is important that the support media is electrically neutral.
Different types of gels which can be used are; Agar and Agarose gel, Starch, Sephadex, Polyacrylamide gels.
PAGE is a subtype of the gel electrophoresis whereby the normal gel is replaced with polyacrylamide gels use as the support matrix.
widely used and has very much importance.
COMPLETE PROCEDURE & USES are described in the slide.
Running of Agarose Gel Electrophoresis Practical Sabahat Ali
Electrophoresis technique used for separation of Macromolecules(DNA, Proteins & their derivatives)
Separation occur on the basis of charge to size ratio
Electrophoresis is a laboratory technique used to separate DNA, RNA, or protein molecules based on their size and electrical charge.
Different types of electrophoresis.
Gel electrophoresis; Agarose Gel electrophoresis; polyacrylamide gel electrophoresis; pulsed-field gel electrophoresis
It is a subtype of the gel electrophoresis whereby the normal gel is replaced with polyacrylamide gels used as support media.
Gels are made by free radical-induced polymerization of acrylamide and N,N’-Methylenebisacrylamide.
It is the most widely used technique of electrophoresis.
This presentation contain the information about gel electrophoresis method , instruments & types.
Electrophoresis is a method through biological molecules are separated by applying an electric field.
Main purpose of this method is to determine the number , amount & mobility of biological component.
There are some internal & external factors that affects the process of electrophoresis.
The bio-molecules have charge on it & when we apply an electric field , the charge particles move to the opposite cathode. In this way, charge particles are separated
There are 3 types of gels that use in this process .
In this buffers are also used which provide ions that carry a current.
Introduction
Gel Electrophoresis
Principle of separation
Instrument and reagents
Factors affecting separation in gel electrophoresis
Applications
Electrophoresis apparatus
Buffer
Power supply
Supporting media
Detection and Quantification
Agarose
Polyacrylamide
Sepration of molecules on the basis of applied Electric Field
Categorized into 1) Zone Electrophoresis 2) Moving Boundary Electrophoresis
We can seprate macromolecules (DNA , RNA, PROTEINS )on the basis of their charge, size shape & molecular weight
Separation is brought about through molecular sieving technique, based on the molecular size of the substances. Gel material acts as a "molecular sieve”.
Gel is a colloid in a solid form (99% is water).
It is important that the support media is electrically neutral.
Different types of gels which can be used are; Agar and Agarose gel, Starch, Sephadex, Polyacrylamide gels.
PAGE is a subtype of the gel electrophoresis whereby the normal gel is replaced with polyacrylamide gels use as the support matrix.
widely used and has very much importance.
COMPLETE PROCEDURE & USES are described in the slide.
Running of Agarose Gel Electrophoresis Practical Sabahat Ali
Electrophoresis technique used for separation of Macromolecules(DNA, Proteins & their derivatives)
Separation occur on the basis of charge to size ratio
Electrophoresis is a laboratory technique used to separate DNA, RNA, or protein molecules based on their size and electrical charge.
Different types of electrophoresis.
Gel electrophoresis; Agarose Gel electrophoresis; polyacrylamide gel electrophoresis; pulsed-field gel electrophoresis
It is a subtype of the gel electrophoresis whereby the normal gel is replaced with polyacrylamide gels used as support media.
Gels are made by free radical-induced polymerization of acrylamide and N,N’-Methylenebisacrylamide.
It is the most widely used technique of electrophoresis.
This presentation contain the information about gel electrophoresis method , instruments & types.
Electrophoresis is a method through biological molecules are separated by applying an electric field.
Main purpose of this method is to determine the number , amount & mobility of biological component.
There are some internal & external factors that affects the process of electrophoresis.
The bio-molecules have charge on it & when we apply an electric field , the charge particles move to the opposite cathode. In this way, charge particles are separated
There are 3 types of gels that use in this process .
In this buffers are also used which provide ions that carry a current.
Introduction
Gel Electrophoresis
Principle of separation
Instrument and reagents
Factors affecting separation in gel electrophoresis
Applications
Electrophoresis apparatus
Buffer
Power supply
Supporting media
Detection and Quantification
Agarose
Polyacrylamide
Sepration of molecules on the basis of applied Electric Field
Categorized into 1) Zone Electrophoresis 2) Moving Boundary Electrophoresis
We can seprate macromolecules (DNA , RNA, PROTEINS )on the basis of their charge, size shape & molecular weight
Gel Electrophoresis:
Definition:
Gel electrophoresis is a laboratory technique used to separate and analyze macromolecules such as DNA, RNA, and proteins based on their size and charge. It is a fundamental tool in molecular biology and biochemistry research.
Principle:
The basic principle involves applying an electric field to a gel matrix, typically made of agarose or polyacrylamide. When a current is applied, charged molecules migrate through the gel at rates determined by their size and charge. Smaller molecules move more quickly through the gel, while larger ones move more slowly.
Applications:
Gel electrophoresis is widely used in various applications, including:
-DNA fingerprinting
-Analysis of PCR products
-Checking the purity of nucleic
acid samples
-Protein analysis and
characterization
What is agarose gel electrophoresis. Its source, history, principle.
Equipment needed for Gel electrophoresis, and protocol.
Applications and pros and cons of Agarose gel electrophoresis
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Scott Malcolm is a great businessman, who lives in Dallas, Taxes. He explains here about biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length and to estimate the size of DNA and RNA fragments.
Agarose gel electrophoresis by KK Sahu sirKAUSHAL SAHU
INTRODUCTION.
HISTORY.
PROCESS OF GEL ELECTROPHORESIS.
AGAROSE GEL ELECTROFORESIS.
POLYACRYALAMIDE GEL ELECTRIPHORESIS.
GEL CONDITION.
DENATURETION.
NATIVE.
BUFFERS.
USES.
CONCLUSION.
REFFERENCES.
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Slide 1: Title Slide
Extrachromosomal Inheritance
Slide 2: Introduction to Extrachromosomal Inheritance
Definition: Extrachromosomal inheritance refers to the transmission of genetic material that is not found within the nucleus.
Key Components: Involves genes located in mitochondria, chloroplasts, and plasmids.
Slide 3: Mitochondrial Inheritance
Mitochondria: Organelles responsible for energy production.
Mitochondrial DNA (mtDNA): Circular DNA molecule found in mitochondria.
Inheritance Pattern: Maternally inherited, meaning it is passed from mothers to all their offspring.
Diseases: Examples include Leber’s hereditary optic neuropathy (LHON) and mitochondrial myopathy.
Slide 4: Chloroplast Inheritance
Chloroplasts: Organelles responsible for photosynthesis in plants.
Chloroplast DNA (cpDNA): Circular DNA molecule found in chloroplasts.
Inheritance Pattern: Often maternally inherited in most plants, but can vary in some species.
Examples: Variegation in plants, where leaf color patterns are determined by chloroplast DNA.
Slide 5: Plasmid Inheritance
Plasmids: Small, circular DNA molecules found in bacteria and some eukaryotes.
Features: Can carry antibiotic resistance genes and can be transferred between cells through processes like conjugation.
Significance: Important in biotechnology for gene cloning and genetic engineering.
Slide 6: Mechanisms of Extrachromosomal Inheritance
Non-Mendelian Patterns: Do not follow Mendel’s laws of inheritance.
Cytoplasmic Segregation: During cell division, organelles like mitochondria and chloroplasts are randomly distributed to daughter cells.
Heteroplasmy: Presence of more than one type of organellar genome within a cell, leading to variation in expression.
Slide 7: Examples of Extrachromosomal Inheritance
Four O’clock Plant (Mirabilis jalapa): Shows variegated leaves due to different cpDNA in leaf cells.
Petite Mutants in Yeast: Result from mutations in mitochondrial DNA affecting respiration.
Slide 8: Importance of Extrachromosomal Inheritance
Evolution: Provides insight into the evolution of eukaryotic cells.
Medicine: Understanding mitochondrial inheritance helps in diagnosing and treating mitochondrial diseases.
Agriculture: Chloroplast inheritance can be used in plant breeding and genetic modification.
Slide 9: Recent Research and Advances
Gene Editing: Techniques like CRISPR-Cas9 are being used to edit mitochondrial and chloroplast DNA.
Therapies: Development of mitochondrial replacement therapy (MRT) for preventing mitochondrial diseases.
Slide 10: Conclusion
Summary: Extrachromosomal inheritance involves the transmission of genetic material outside the nucleus and plays a crucial role in genetics, medicine, and biotechnology.
Future Directions: Continued research and technological advancements hold promise for new treatments and applications.
Slide 11: Questions and Discussion
Invite Audience: Open the floor for any questions or further discussion on the topic.
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2. Why electrophoresis?
• To separate DNA fragments from each other
• To determine the sizes of DNA fragments
• To determine the presence or amount of DNA
• To analyze restriction digestion products
3. Introduction Of Agarose Gel
Electrophoresis
• Agarose gel electrophorresis is a method
to separate DNA or RNA molecules by size.
• This is achieved by moving negatively charged
nucleic acid molecules through an agarose
matrix with an electric field (electrophoresis).
• Shorter molecules move faster and migrate
faster than longer ones .
4. Principle of electrophoresis
• powerful separation method frequently used to
analyze DNA fragments generated by restriction
enzymes
• convenient analytical method for determining
the size of DNA molecules in the range of 500 to
30,000 base pairs.
• employs electromotive force to move molecules
through a porous gel
5. Principle (cont.)
• separates molecules from each other on
the basis of
–size and/or
–charge and/or
–shape
• basis of separation depends on how the
sample and gel are prepared
8. Agarose
• A linear carbohydrate polymer extracted from
seaweed , agarobiose
• forms a porous matrix as it gels
– shifts from random coil in solution to structure in
which chains are bundled into double helices
9. agarose
•Basic unit of agar which is a cell wall and intercellular component of some
red marine algae, usually Gelidium and Gracillaria.
•Linear polysaccharide that contains double helices stabilized by water
molecules.
•Exterior hydroxyl groups allow helices to aggregate
into suprafibers that branch off to form a matrix.
10. *AGAROSE GEL
A highly purified uncharged polysaccharide derived from
agar.
Used to separate macromolecules such as nucleic acids,
large proteins and protein complexes.
It is prepared by dissolving 0.5% agarose in boiling water
and allowing it to cool to 40°C.
It is fragile because of the formation of weak hydrogen
bonds and hydrophobic bonds.
11. TYPES OF AGAROSE
• Standard Agarose - LE
Gels at 35-38oC; Melts at 90-95oC
Becomes opaque at high concentrations
• Low Melting Agarose (NuSieve)
Gels at 35oC; Melts at 65oC
Often used to isolate DNA fragments
from gel
Intermediate forms or combinations of LE
and NuSieve can provide sturdy, translucent
gels at high agarose concentrations .
13. Gel Casting Trays
• available in a variety of
sizes and composed of
UV-transparent plastic.
• The open ends of the
trays are closed with
tape while the gel is
being cast, then
removed prior to
electrophoresis.
14. Applied voltage
• voltage, rate of migration
• The higher the voltage, the more quickly the
gel runs
• But if voltage is too high, gel melts
• The best separation will apply voltage at no
more than 5V/cm of gel length.
15. Buffers
• During electrophoresis water undergoes
hydrolysis : H2O H + OH-
• Buffers prevent the pH from changing by
reacting with the H+ or OH- products
• Most common buffer used is called TRIS
– [tris(hydroxymethyl)aminomethane]
16. Buffers (cont.)
• Another compound is added to make Tris an
effective buffer — either boric or acetic acid
• Another compound is added to bind metals
EDTA
• The buffer is either TBE or TAE
TBE is made with Tris/Boric Acid/EDTA
TAE is made with Tris/Acetic Acid/ EDTA
17. Staining of DNA
• To make DNA fragments visible after
electrophoresis, the DNA must be stained
• The favorite—ethidium bromide
• When bound to DNA it fluoresces under
ultraviolet light (reddish –orange colour)
• Convenient because it can be added directly
to the gel
• Sensitive—detects 0.1ug of DNA
18. *Dye DNA and place into gel
The gel is made out of
agarose, which is similar
to jello.
The gel is made with
wells at one end so
that the DNA can be
loaded into the gel.
19. Ethidium bromide
• The standard concentration
used in staining DNA in gels is
0.5-1ug/mL
• Ethidium bromide is a
fluorescent dye that
intercalates between bases of
nucleic acids and allows very
convenient detection of DNA
fragments in gels.
• Inserting itself between the
base pairs in the double helix
20. Staining of DNA (cont.)
• UV absorbance maxima at 300 and 360 nm and
emission maxima at 590 nm.
• Detection limit of bound DNA is 0.5-5 ng/band.
• ethidium bromide is mutagenic so care must be
taken while handling the dye.
• Othe alternatives for ethidium bromide :
Methylene blue
Syber safe
xylene cyanol
bromphenol blue
21. A Comb
• A comb is placed in the
liquid agarose after it
has been poured
• Removing the comb
from the hardened gel
produces a series of
wells used to load the
DNA
22. DNA ladder
• It is a solution of DNA
molecules of different length
• DNA Ladder consists of known
DNA sizes used to determine
the size of an unknown DNA
sample.
• The DNA ladder usually
contains regularly spaced sized
samples which when run on an
agarose gel looks like a
"ladder".
24. Method For Electrophoresis
Add running buffer, load samples and marker
Run gel at constant voltage until band separation occurs
Pour into casting tray with comb and allow to solidify
View DNA on UV light box and show results
Prepare agarose gel
Melt, cool and add Ethidium Bromide. Mix thoroughly.
25. • DNA is negatively charged.
+-
Power
DNA
• When placed in an electrical field, DNA will migrate toward the
positive pole (anode).
H
O2
• An agarose gel is used to slow the movement of DNA and separate
by size.
26. +-
Power
DNA
How fast will the DNA migrate?
strength of the electrical field, buffer, density of agarose gel…
Size of the DNA!
*Small DNA move faster than large DNA
…gel electrophoresis separates DNA according to size
small
large
27. • Within an agarose gel, linear DNA migrate
inversely proportional to the log10 of their
molecular weight.
28. *Smaller pieces of DNA travel farther than Larger pieces of
DNA
End result!
29. applications
• Solve criminal cases
• Solve paternity cases
• Diagnose genetic diseases
• Determine genetic kinship among species
30. applications
• Gel electrophoresis is commonly used in plant
breeding and genomics for genotyping
with molecular markers,
• specific DNA fragments used as markers and
isolated from individual plants are amplified
by the polymerase chain reaction (PCR) and
the resulting DNA fragments are subsequently
loaded on a gel.