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.
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.
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.
Centrifugation principle and types by Dr. Anurag YadavDr Anurag Yadav
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basic Principle
centrifugal force
types of centrifugation based on use and rotor type
application of the each type of centrifuge
Ultracentrifuge in detail
application in general
Gel electrophoresis native, denaturing&reducingLovnish Thakur
Electrophoresis is a technique used to separate and sometimes purify macromolecules - especially proteins and nucleic acids - that differ in size, charge or conformation.
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The following presentation contains helpful information regarding SDS-PAGE, including the history, introduction, principle, instrumentation, advantages and applications of SDS-PAGE.
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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.
Centrifugation principle and types by Dr. Anurag YadavDr Anurag Yadav
concept of cnetrifugation,
basic Principle
centrifugal force
types of centrifugation based on use and rotor type
application of the each type of centrifuge
Ultracentrifuge in detail
application in general
Gel electrophoresis native, denaturing&reducingLovnish Thakur
Electrophoresis is a technique used to separate and sometimes purify macromolecules - especially proteins and nucleic acids - that differ in size, charge or conformation.
Introduction, Principle, Instrumentation and Applications of SDS-PAGEMohammed Mubeen
The following presentation contains helpful information regarding SDS-PAGE, including the history, introduction, principle, instrumentation, advantages and applications of SDS-PAGE.
GENE CLONING,ITS HISTORY, NEW ADVENT IN GENE CLONING, PCR IMPORTANCE ,APPLICATION OF GENE CLONING,STEPS OF GENE CLONING,Antisense technology,Gene cloning in agriculture,Somatic cell therapy,Role of gene cloning in identification of genes responsible for human diseases,Synthesis of other recombinant human proteins and recombinant vaccines
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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.
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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
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Electrophoresis is a general term that describes the migration and separation of charged particles under the influence of an electric field.
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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.
Electro- = flow of electricity,
-phoresis = to carry across
Electrophoresis is a technique used to separate and sometimes purify macromolecules - especially proteins and nucleic acids - that differ in size, charge or conformation. As such, it is one of the most widely-used techniques in biochemistry and molecular biology
Gel electrophoresis is a procedure that separates molecules on the basis of their rate of movement through a gel under the influence of an electrical field.
Methods & Types
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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
7. Material required for agarose gel
electrophoresis
Electrophoresis chamber
Agarose gel
Gel casting tray
Buffer
Staining agent (dye)
A comb
DNA ladder
Sample to be separate
9. What is 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
10. TYPES OF AGAROSE
• Standard Agarose - LE
Gels at 35-38o
C; Melts at 90-95o
C
Becomes opaque at high concentrations
• Low Melting Agarose (NuSieve)
Gels at 35o
C; Melts at 65o
C
Often used to isolate DNA fragments
from gel
Intermediate forms or combinations of LE
and NuSieve can provide sturdy,
translucent gels at high agarose
12. 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.
13. 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.
14. 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]
15. 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
16. 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
17. 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
18. 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
19. 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
20. 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".
22. Method For Electrophoresis
Add running buffer, load samples and markerAdd running buffer, load samples and marker
Run gel at constant voltage until band separation occursRun gel at constant voltage until band separation occurs
Pour into casting tray with comb and allow to solidifyPour into casting tray with comb and allow to solidify
View DNA on UV light box and show resultsView DNA on UV light box and show results
Prepare agarose gel
Melt, cool and add Ethidium Bromide. Mix thoroughly.
Prepare agarose gel
Melt, cool and add Ethidium Bromide. Mix thoroughly.
23. • 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.
24. +-
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
25. • Within an agarose gel, linear DNA migrate
inversely proportional to the log10 of their
molecular weight.