3. WHAT IS GEL ELECTROPHORESIS
Electrophoresis is the term used for the
procedure where under the influence of
voltage, a charged particle moves.
It is a standard method for separation,
identification, analysis and purification of:
DNA molecules
protein molecules
4.
5. Electrophoresis consists of the migration
of a charged molecules under the
influence of electric field (from negative
to positive).
A buffer solution is use to conduct
electricity through the whole setup of the
gel electrophoresis.
The molecule will migrate through the gel
depending upon the size and shape.
6. Gel electrophoresis is used:
Forensics
Molecular biology
Genetics
Microbiology
Biochemistry
The results can be analyzed quantitatively by
visualizing the gel with UV light and a gel imaging
device; analyzing the intensity of the band or the
measure of the spot of interest.
8. AGAROSE GEL ELECTROPHORESIS
Agarose is a linear polymer extracted from
seaweed that forms a gel matrix by hydrogen-
bonding when heated in a buffer and allowed
to cool.
The agarose gel is used to separate DNA and
RNA fragments.
Agarose gels separate DNA fragments differing
by a hundred or more base pairs.
9. DNA has negative charge so it
migrates towards the positive end.
This is due to its double helical
physical structure, which contains a
phosphate backbone.
10. The density and porosity of the gel matrix is
determined by the concentration of agarose
used.
The grater the agarose concentration, the
smaller the pores created in the gel matrix,
the more difficult it is for larger DNA
molecules to move through.
Agarose % Optimum Resolution for DNA
0.5 1,000-30,000bp
0.7 800-12,000bp
1.0 500-10,000bp
1.2 400-7,000bp
1.5 200-3,000bp
2.0 50-2,000bp
11.
12.
13. POLYACRYLAMIDE GEL
ELECTROPHORESIS
Like Agarose Gels, Polyacrylamide gels are
used to separate protein molecules by
shape, size and charge.
Polyacrylamide is a polymer of acrylamide
monomers.
14. Polyacrylamide is specifically used for
proteins because it provides the protein
with an environment where it will not
become denatured.
Allowing different sized proteins to
move at different rates.
15. Since we are trying to separate many
different protein molecules of a variety of
shapes and sizes, we first want to get them to
be linear so that the proteins no longer have
any secondary, tertiary or quaternary
structure.
16. To have proteins with linear structures we
use sodium dodecyl sulfate (SDS).
SDS is a detergent that can dissolve
hydrophobic molecules, resulting in
proteins with linear structures.
17. Another problem we face with proteins is that
they do not have a specific charge.
This is another reason why SDS is important.
SDS has a negative charge and by dissolving
the protein in it, the protein becomes
negatively charged.
Allowing it to run properly through the gel
(from negative to positive).
18. Get your sample
obtained from
previous purifying
technique (i.e. PCR)
Set up gel, remove
comb
Load Buffer
Load Sample
Run Gel
Stain and look at with
UV light
19.
20. APPLICATIONS OF GELS:
Estimation of the size of DNA and
protein molecules.
Analysis of PCR products, i.e. in
molecular genetic diagnosis or genetic
fingerprinting
Separation of restricted genomic DNA or
of RNA.
