I AM HAFIZ MUHAMMAD WASEEM from mailsi vehari BSc from science college Multan MSC university of education Lahore i love Pakistan and my teachers

1. HAFIZ
MUHAM
MAD
WASEEM
BA BA G
LAHORE
Micrometry

2. Advanced Analytical Techniques
UNIVESITY OF EDUCATION
LAHORE PAKISTAN

3. Introduction
Gel electrophoresis?
 Agarose is a polysaccharide
made from seaweed
 It is dissolved in buffer,
heated and cooled to a
gelatinous solid in the form
of inert matrix
 A gel is a colloid, suspension
of tiny particles in a medium,
occurring in a solid form (like
gelatin)
 Gel electrophoresis refers to
the separation of charged
molecules like nucleic acids,
proteins, etc. when an electric
current is applied
 It is the easiest analyzing
macromolecules

4. Introduction
How GE works?
 Frictional force of the gel
matrix acts as a molecular
sieve which separate the
molecules on the basis of their
size
 Macromolecules are forced to
move through the pores when
electrical current is applied
 Most agarose gels are made
between 0.7% and 2% of
agarose in some suitable
buffer
 The rate of migration through
the electric field depends on
• Charge of the molecules
• Size of the molecules
• Shape of the molecules

5. Introduction
How GE works?
 After staining, the separated
macromolecules in the gel
are seen in the form of bands
 A standard is also run in one
lane
 It is most commonly used
technique in biochemistry
and molecular biology

6. Introduction
Types of GE
 Polyacrylamide gel
electrophoresis (PAGE) is used
for separation of proteins
ranging in size from 5 to
2,000 kDa
 It uses sodium dodecyl sulfate
(SDS) as detergent which
denature the proteins and
enable them to move
independently of each other
 Agarose gel electrophoresis
uses agarose
 Pores of an agarose gel are
large so it is used to separate
macromolecules such as
• Nucleic acids
• Large proteins
• Protein complexes

7. Advanced Analytical Techniques

8.  Most agarose gels are made
between 0.7% and 3%
 Low percentage gels are very
fragile and are used to
separate DNA molecules from
5 to 10 kb in size
 While high percentage gels
are used to separate smaller
molecules
 DNA and RNA molecules
(negatively charged) move
towards anode when electric
current is applied
 Separation depends upon
composition and ionic
strength of buffers used
Properties of AGE

9.  Higher the voltage used,
faster the nucleic acids will
separate
 Visualization of the nucleic
acids may be achieved by
ethidium bromide, silver
staining, etc.
 3-D structure of agarose gels
is held together with
hydrogen bonds
 DNA bands can be cut from
the gel and processed further
for the research
 Buffers used contain EDTA to
inactivate many nucleases

10. Advanced Analytical Techniques

11. Requirements of AGE
Gel Apparatus
 A power supply, a gel
chamber, combs,
micropipettes, ladder,
buffers, dyes, UV illuminator,
electrodes, cables, gel
mixtures, gel doc system, are
needed

12. Requirements of AGE
Buffers
 Buffers maintain a pH either
by absorbing or releasing
Hydrogen ions
 Buffers - either TBE or TAE
provide ions to ensure
electrical conductivity along
with other functions
 Not only agarose is dissolved
in the buffers but the gel slab
is also submerged in the
buffers after solidifying
 TAE has the minimum
buffering capacity but has the
best resolution as compared
with other buffers

13. Requirements of AGE
DNA Ladder
 One of the wells in the gel is
loaded with a DNA ladder
 This is used as a marker to
compare the separated DNA
fragments in the form of
bands in the gel and to
estimate their sizes

14. Requirements of AGE
Visualizing DNA
 Ethidium bromide (EtBr), a
fluorescent dye which is
visualized when excited by
UV light is generally used
 Gel is soaked in a solution of
EtBr and the DNA bands take
up the dye
 Then the gel is placed under
UV illuminator and visualized
and photographed for
further analysis

15. Advanced Analytical Techniques

16.  Gel is prepared by dissolving
the agarose powder as per
requirement in the suitable
buffer
 It is heated so that agarose is
melted in the buffer
 The melted agarose is allowed
to cool before pouring the
solution into a cast
 A comb is placed in the cast to
create wells for loading the
samples
 Place the gel at 4C or at room
temperature till it is
completely solidified
Procedure of AGE
Casting of gel

17.  Once the gel is solidified, the
comb is removed for loading
DNA samples
 Loading buffer is mixed with
the samples to increase
density and to add some dye
(bromophenol blue) to the
samples
 The gel is placed in
electrophoresis unit and is
covered with the buffer
 Load ladder and samples in
the wells
 Electrophoresis is carried at
the suitable voltage
Procedure of AGE
Loading of samples

18.  Electrophoresis is carried out
horizontally or vertically
 Buffer used in the gel is the
same as the running buffer in
the electrophoretic tank
 Run the gel at 80-150 volatge
until the dye is about 75-80%
down the gel
 Afterwards, the gel is
removed from the gel box
 DNA fragments are visualized
on placing the gel under UV
source
 Size of DNA fragments is
estimated with the help of
ladder
Procedure of AGE
Electrophoresis

19. Advanced Analytical Techniques

20.  It is used to analyze DNA
molecules which are cut by
restriction enzymes
 Cut fragments can be used
for cloning purposes
 Analysis of PCR products, e.g.
in molecular diagnostics
or genetic fingerprinting
 Separation of DNA fragments
for extraction and
purification purposes
 Separation of restricted
genomic DNA prior
to Southern transfer or of
RNA before Northern
transfer
Applications of AGE

21.  DNA molecules can be easily
recovered from the gels
without any damage
 It is also used for screening
protein abnormalities in
various biological fluids like
serum, urine, CSF, etc.
 Size of DNA molecules can
be analyzed by lambda DNA
ladder
 Two dimensional
electrophoresis is useful for
identifying a particular
protein from thousands of
other proteins between
control and treated samples
Applications of AGE