Gel electrophoresis is a method to separate macromolecules like DNA, RNA, and proteins based on their size and charge by applying an electric current through a gel matrix. Most molecular biology labs use agarose gel electrophoresis to separate and analyze DNA fragments. During electrophoresis, DNA molecules are separated based on their rate of migration through the agarose gel under an applied electric field with smaller fragments moving faster.

2. Electrophoresis
The Separation of macromolecules under the influence of
a uniform electric field through a matrix which is porous
in nature is to be termed as ELECTROPHORESIS

3. Electrophoresis
Zone
Electrophoresis
Paper
electrophoresis
Gel
electrophoresis
Isoelectric
foucusing
Immunoelectroph
oresis
 Different types of electrophoresis

4. • Gel electrophoresis is a method for separation and
analysis of macromolecules (DNA, RNA and proteins)
and their fragments, based on their size and charge.
• Gel electrophoresis is a widely used technique for
the analysis of nucleic acids and proteins.
• Most every molecular biology research laboratory
routinely uses agarose gel electrophoresis for the
preparation and analysis of DNA.

5. Agarose
Detector

6. •Employs electromotive force to move
molecules through a porous gel
•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. Gel electrophoresis
Horizontal gel
electrophoresis
Vertical gel
electrophoresis
 Important types of gel electrophoresi

8. Porous
Material
Proteins Entering
Porous Material
Smallest Move
Fastest
•Also called Agarose gel electrophoresis
•In this gel electrophoresis the matrix used is a gel and is
made up of agarose
•Agarose – a complex sugar chain from red seaweed. It
has a large pore size good for separating large molecules
quickly.
Horizontal Gel Electrophoresis

9. Components of an Electrophoresis System
Power supply and chamber, a source of negatively
charged particles with a cathode and anode
Buffer, a fluid mixture of water and ions
Agarose gel, a porous material that DNA migrates
through
Gel casting materials
DNA ladder, mixture of DNA fragments of known
lengths
Loading dye, contains a dense material and allows
visualization of DNA migration
DNA Stain, allows visualizations of DNA fragments
after electrophoresis

10. Buffer
Dyes Power Supply
+
-
Agarose
gel
Cathode
Anode

11. Where does the current come from?
 A direct current power supply
 Ions supplied by the buffer
 The charge on the macromolecules being
separated
 Electrolysis of water

12.  Electrolysis of water
 4H2O  2H2 + O2 + 2H2O
 self-ionization of water throughout
the buffer: 4H20  4H+ + 4OH-
 At the negative pole
 4H+ + 4e-  2H2
 At the positive pole
 4OH-  O2 + 2H2O + 4e-

13. What factors affect mobility of linear ds
DNA?
 Pore size of the gel
  [agarose]   pore size
  pore size   friction   mobility
 Voltage across the gel
  voltage   mobility
 Length of the DNA molecule
 smaller molecules generate less friction and so
move faster
 Ethidium bromide (stain) intercalated into DNA
 decreases charge to mass ratio and so decreases
mobility

14. General procedure
1. Casting of gel
2. Loading of gel sample
3. Electrophoresis
4. Staining and visualization
5. Downstream procedure

15. Factors affecting resolution
 Resolution = separation of fragments
 The “higher” the resolution, the more space between
fragments of similar, but different, lengths
 Resolution is affected by
 agarose type
 agarose concentration
 salt concentration of buffer or sample
 amount of DNA loaded in the sample
 voltage

16.  Linear carbohydrate polymer extracted from red
seaweed , agarbiose
 forms a porous matrix as it gels
 shifts from random coil in solution to structure in
which chains are bundled into double helices
What is Agarose ?

17. % Agarose
(w/v)
Size Range (kb prs) for
Optimal Separation
0.5 2-30
0.75 0.7-20
1.0 0.5-10
1.5 0.2-3
2.0 0.1-2
Resolution of ds linear DNA fragments in
agarose gels
1. 1%gels are common for many applications.
2. Up to 3% can be used for separating very tiny fragments but a
vertical polyacrylamide gel is more appropriate in this case

18. Buffer Systems
 Remember, buffer systems include weak acids and/or
bases that do not dissociate completely.
 If ions resulting from dissociation are “removed,”
more weak acid and/or base will dissociate.
 Purposes of buffer
 Keep solution at pH compatible with molecules being
separated
 Generate ions consistently to
 maintain current
 keep resistance low
 Both gel and the solution in the gel box are buffered.

19. Buffer Systems (cont’d)
 Two commonly used buffers for routine agarose gel
electrophoresis
 TAE, pH 8.0, ~50 mM - Tris, Acetate, EDTA
 TBE, pH 8.0, ~50 mM - Tris, Borate, EDTA
 Tris (T) is a weak base.
 Acetic (A) acid and boric (B) acid are weak acids.
 Acetic acid is more completely ionized at pH 8.0 than
is boric acid, so TBE has a high buffer capacity than
TAE.

20. Non-denaturing agarose gel loading solutions
 Composition
 tracking dyes
 are used to follow progress of
electrophoresis
 sometimes interfere with
later visualization of DNA
 a solute to increase density
 so that sample falls to bottom
of loading well with minimal
dilution
 solute examples: glycerol,
Ficoll
 Other gel types, with different
purposes, use different loading
solutions!

21. Voltage
  voltage,  rate of migration
 to increase the voltage
 increase the setting on the power supply
 increase the resistance
 decrease the gel thickness
 decrease the ion concentration
 if voltage is too high, gel melts
 as voltage is increased, large molecules migrate at a rate
proportionally faster than small molecules, so
 lower voltages are better for resolving large fragments
 but the larger ds DNA fragments are always slower than
the smaller ones

22. Ethidium bromide staining
 Binds to DNA by intercalation between stacked bases
 lies perpendicular to helical axis
 makes Van der Waals contacts with bases above and below
 Allows DNA visualization after gel electrophoresis
 EtBr intercalates with DNA and fluoresce under ultraviolet light
thereby allowing DNA visualization after Gel Electrophoresiswhile
 Proteins may be visualised using silver stain or Coomassie
Brilliant Blue

23.  Agarose Gel Electrophoresis :OVERVIEW