3. Invention
of electrophoresis
• During the 1930s Arne Tiselius developed a method
which makes use of this phenomenon to separate
different substance from one another.
• Electrophoresis was successfully used to separate
DNA and RNA samples beginning in the 1960s.
Initially, agar, a natural carbohydrate, was used as a
separation medium for electrophoresis, but this was
replaced in the late 1960s by agarose, a
polysaccharide which is one of the main
components of agar.
4. Principle of
Gel Electrophoresis
• Charged molecules move through a gel
when an electric current is passed across
it.
• An electric current is applied across the
gel so that one end of the gel has a
positive charge and the other end has a
negative charge.
• The movement of charged molecules is
called migration. Molecules migrate
towards the opposite charge.
5. Apparatus
• Agarose, flask, buffer, gel mold, gel comb,
microvawe, electrophoresis box ,micropipette,
pipette tips, loading buffer, DNA sample, DNA
standard size, power supply, stain, UV light box.
This Photo by Unknown author is licensed under CC BY.
6. Steps in the
process
1. Make the gel.
2. Set up the Gel
apparatus.
3. Load the DNA
sample into the
gel.
4. Hook up the
electrical current
and run the gel.
5. Stain the gel
and analyze the
result.
7. Gel Preparation
• Add a gel (Starch gel, Agarose gel, Polyacrylamide gel ) in flask
• Add buffer
• Heat the mixture
• Pour the melted mixture in the mold
• Place the comb
8. Set up the gel
apparatus
• Pour the buffer in electrophoresis box
• Place the gel (still in the mold) inside box
• Gel should be just barely submerged in
the buffer
• Buffer will conduct electrical charge and
prevent gel from drying.
9. Load the DNA sample
into the gel
• Add loading buffer (dye that make sample
easy to see and thicker) in DNA sample using
Micropipette.
• Transfer DNA sample from the tube into the
well of gel using micropippette.
• Using a clean pipet tip transfer DNA size
standard (DNA strand of known length) into
the well next to sample.
10. Hook up the electrical
current and run the gel
• Negative charge is connected near well
and positive charge at other end.
• Turn on power supply ( Check the air
bubble coming out of the electrode at
both ends.)
• Repelled by negative charge the DNA
moves through the gel towards positive
charge end of the gel.
11. Stain the gel and analyze the result
• Drag the gel out of the mold and put into the
DNA staining solution (Ethidium bromide- binds
to DNA in between the rungs)
• It can damage DNA in your cells so wear gloves
and avoid direct contact with staining solution.
• Place the gel on UV light box and analyze.
• for visualizing DNA following dyes can also be
used
• SYBR Gold dye can be used to stain double or
single-stranded DNA or to stain RNA.
• SYBR Green.
• SYBR Safe.
• Eva Green.
12. Application
• Estimation of the size of DNA molecules following
restriction enzyme digestion, e.g. in restriction
mapping of cloned DNA.
• Analysis of PCR products, e.g. in molecular genetic
diagnosis or genetic fingerprinting
• Separation of restricted genomic DNA prior to
Southern transfer, or of RNA prior to Northern
transfer.
• Gel electrophoresis is used in forensics, molecular
biology, genetics, microbiology and biochemistry.
13. • Protein and
Antibody
Interactions
• Another common form of electrophoresis is
immunoelectrophoresis, which analyzes the presence and
behaviors of certain proteins. Many medical conditions,
including multiple sclerosis, kidney disease and some
cancers result in the creation of abnormal protein
molecules. These can be detected by performing
electrophoresis on urine or blood samples and watching for
any variance from the normal quantities and types of
protein. Immunoelectrophoresis can also be used to detect
specific proteins called immunoglobulins, which act as
antibodies. These are part of the body's immune system and
attack foreign proteins, such as viruses or allergens.
Analyzing these antibodies can help identify new therapies
to treat those invaders and also provides insight into
conditions such as allergies and autoimmune disorders,
which can result from malfunctioning antibodies.
14. Testing Antibiotics
• Electrophoresis plays a number of roles in the testing of
antibiotics. One of the most common is testing the purity of
an antibiotic. By applying electrophoresis to a solution
containing the antibiotic in the form of a paper strip
impregnated with the antibiotic or a capillary – a very thin
tube – filled with the solution, researchers can differentiate
between the antibiotic itself and any impurities. They can
also determine how concentrated the antibiotic is, which is
crucial for applying accurate dosages. Antibiotic research
extends into the realm of genetic testing, identifying genes
that might indicate resistance to specific antibiotics
15. Testing Vaccines
• Electrophoresis is useful in both the creation
and production of vaccines. The purpose of a
vaccine is to help the body generate antibodies
to a potentially dangerous pathogen, and
electrophoresis is a useful method for detecting
those antibodies. Researchers can use the
technique to compare the effect of a vaccine or
multiple versions of a vaccine across a large
number of test subjects or other variables.
Once a vaccine is in production, electrophoresis
also provides a quick and effective way of
testing production batches for consistency and
purity.
16. Common errors in
electrophoresis
• Sample contamination. Whatever you
are measuring, the first step to get
accurate results is an uncontaminated
sample.
• Problems in the gel. Many errors are due
to problems with the gel.
• Load of incorrect samples.
• Problems in the electric current.
• Problems in visualization.
