Gel electrophoresis

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

1. DNA
ELECTRO-
PHORESIS

2. Self Intro!
FATIMA ASLAM KHAN
PL01
[email protected]

3. Biomolecules SeparationTechniques
Chromato
graphy
Electro
phoresis
Centri
fugation
01 02 03

4. Electrophoresis
Separation technique
01

5. Electrophoresis
•Electro = flow of electricity,
•Phoresis= to carry across
•Electrophoresis is a technique
commonly used in the lab to
separate charged molecules,
like DNA, according to size.
•Is a separation method that is
based on the migration of
charged species in a supporting
medium (a liquid or a
hydrophilic gel) under the
influence of an electric field

6. Principle
•An electrophoretic system
consists of two electrodes of
opposite charge (anode,
cathode), connected by a
conducting medium called an
electrolyte.
•The separation effect on the
ionic particles results from
differences in their velocity (v),
which is the product of the
particle's mobility(m) and the
field strength (E):V=Me

7. Types of Electrophoresis
Moving
Boundary
Electro
phoresis
Zone
Electro
phoresis
01 02

8. Zone Electrophoresis
Paper
Electrophoresis
Gel Electro
phoresis
Thin Layer
Electrophor
esis
Cellulose
Bounding
Electrophoresis
01 02 03 04

9. Moving Boundary Electrophoresis
Capillary
Electrophoresis
Isotacho
phoresis
Isoelectric
Focusing
Immuno
Electrophoresis
01 02 03 04

10. Gel Electrophoresis
Electrophoresis involvesrunning a current
through agel containing the moleculesof
interest.
Based on their sizeand charge, the
moleculeswill travel through the gel
indifferent directions or atdifferent
speeds, allowingthem to be separated
fromone another.
Gel electrophoresisis a
technique used to separate
DNA fragments (or other
macromolecules, such as
RNA and proteins) based on
their size and charge.
What's it? How?

11. What is a Gel?
●Gel electrophoresis involves a gel: a slab of Jello-like material.
●Gel is a colloid in a solid form (99% is water).
●Gels for DNA separation are often made out of a polysaccharide calledagarose,
which comes as dry, powdered flakes.
●When the agarose is heated in a buffer (water with some salts in it) and allowed to
cool, it will form a solid, slightly squishy gel.
●At the molecular level, the gel is a matrix of agarose molecules that are held
together by hydrogen bonds and form tiny pores.
At one end, the gel has pocket-like indentations calledwells, which are where
theDNA samples will be placed.
●Before the DNA samples are added, the gel must be placed in agel box.

12. What is a Gel?

13. What is a Gel?
●One endof the box is hooked to a positive electrode, while the other end is
hookedto a negative electrode.
●The main body of the box, where the gel is placed, is filledwith a salt-containing
buffer solution that can conduct current.Thebuffer fills the gel box to a level where
it just barely covers the gel.
●The end of the gel with the wells is positioned towards the negative electrode.
●The end without wells (towards which the DNA fragments will migrate)
ispositioned towards the positive electrode..

14. Types of Gels Matrix Used
Poly acryl amideAgarose
01 02
•Large pore size gel will
be made
•Gel casted horizontally
•Used to separate DNA ,
RNA
•Staining can be done
before pouring the gel
•Smallpore size gel will be
made
•Gel casted vertically
•Used to separateProtein&
Small size DNA >1000 bp
•Staining can be doneafter
pouring the gel

15. Agarose Gels
•Concentration –1% -3%
•Separation in agarose gels is achieved due to
size(MW) of DNA because of resistance to their
movement caused by the gel matrix
•Agarose dissolves when added to boiling liquid and
forms gels when temperature is lowered to 40°C
•Staining is done by ethidium bromide
SYBR green and methylene blue
•Agarose is a linear polysaccharide :
Agarobiosecomprises alternating
units of galactose and 1,3-
anhydrogalactose
•Commomlyused,less
expensive,providesgood separation
What's it? How Much?

17. Poly acryl amide Gels
•It is prepared by polymerizing acryl amide monomers in the presence of
methylene-bis-acrylamide to cross link the monomers.
•Structure of acrylamide (CH=CH-CO-NH)
•Polyacrylamide gel structure held together by covalent cross-links.
•Polyacrylamide gels are tougher than agarose gels.
'It is thermostable, transparent, strong and relatively chemically inert.
•Gels are uncharged and are prepared in a variety of pore sizes.
•Proteins are separated on the basis of charge to mass ratio and molecular
size

18. Buffer Used
•Buffers are said to be a mixture of
weak acid and its conjugate base as
well as vice versa.
•As buffers, they also have a
fairlyconstant pH & are able to
conductelectricity because of
theirconcentration of hydrogen
ions
What's it?

19. Buffer Used
•Tris acetate EDTA (TAE) and tris borate EDTA (TBE) are two most
common running buffers used in nucleic acid electrophoresis.
•Agarose gel electrophoresis of the DNA and RNA is routinely
performed using the buffers containing either Tris, acetate and EDTA
(TAE) or Tris, borate and EDTA (TBE).
•TAE also works better for cloning, because TBE contains borate.
Borate in TBE is an inhibitor for many kind of enzymes, such as ligase.
TAE works better for performing the DNA extraction from agarose gel.
Types of Buffers

20. Stains Used
•Ethidium bromide (EtBr) is used for DNA staningto observe DNA in a
gel. EtBr binds between the double helix and gives bright orange
colourwhen observed under UV light.
•The separated fragments in a mixture can be visualized by ethidium
bromide staining followed by UV exposure. Ethidium bromide is added
in the agarose gel once agarose powder is dissolved in buffer and
mixed thoroughly.
•The stain gets dispersed throughout the gel. Ethidium bromide acts as
an intercalating molecule.
•It gets inserted within the species present between the double helix of
DNA.
•This absorbs wavelength corresponding to ultraviolet radiation and
emits another wavelength that is visible to us.

21. Stains Used

22. •DNA is an organic acid, and is negatively charged
(remember, DNA for Negative)
•When the DNA is exposed to an electrical field,
the particles migrate toward the positive electrode
•Smaller pieces of DNA can travel further in a given
time than larger pieces
Working
How do DNA fragments move through the gel?

23. Steps in running a gel
How do DNA fragments move through the gel?
•DNA is prepared by digestion with restriction enzymes
•The gel chamber is set up, the 'comb' is inserted
•The agarose may have a DNA 'dye' added (or it may be
stained later). The agarose is poured onto the gel block
and cooled
•The comb is removed, leaving little 'wells' and buffer is
poured over the gel to cover it completely
•The DNA samples are mixed with a dense loading dye so
they sink into their wells and can be seen

24. Steps in running a gel
How do DNA fragments move through the gel?
•The DNA samples are put in the wells with a
micropipette(Micropipettes have disposable tips and can
accuratelymeasure1/1,000,000 of a litre)
•The power source is turned on and the gel is run.
•The time of the run depends upon the amount of current and % gel
•At the end of the run the gel is removed (it is actually quite stiff)
•The gel is then visualized
•UV light causes the bands of DNA to fluoresce
•The DNA band of interest can be cut out of the geland the DNA
extracted -Or DNA can be removed from the gel by
SouthernBlotting

26. Advantages of
Agarose Gel
Electrophoresis
•Easy to prepare and small concentration of agar is required.
•Resolution is superior to that of filter paper.
•Large quantities of proteins can be separated and recovered.
•Adsorption of negatively charged protein molecule is Negligible.
•It adsorbs proteins relatively less when compared to other
medium.
•Sharp zones are obtained due to less adsorption.
•Recovery of protein is good, good method for preparative purpose

27. Disadvantages of
Agarose Gel
Electrophoresis
•Electro osmosis is high.
•Resolution is less compared to
polyacrylamide gels.
•Different sources and batches of
agar tend to give different results
and purification is often necessary.

28. Applications
•Widely used in Immunoelectrophoresis.
•To separate different types of protein mixtures as well as nucleic acids.
•Used for estimation of molecular weight of proteins and nucleic acids.
•Determination of subunit structure of proteins.
•Purification of isolated proteins.
•Monitoring changes of protein content in body fluids.
•Identifying disulfide bonds between protein
•Quantifying proteins
•Blotting applications

29. DNA Electrophoresis Video Link
DNA Electrophoresis

30. DNA Electrophoresis
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