Electrophoresis as a technique for qualitative analysis of nucleic acids and proteins is highly dependable.

1. • Electrophoresis is a method whereby charged
molecules in solution, chiefly proteins and nucleic
acids, migrate in response to an electrical field.
• Their rate of migration through the electrical field,
depends on the strength of the field, on the net charge,
size, and shape of the molecules, and also on the ionic
strength, viscosity, and temperature of the medium in
which the molecules are moving.
• As an analytical tool, electrophoresis is simple, rapid
and highly sensitive.
• It can be used to study the properties of a single
charged species or mixtures of molecules. It can also
be used preparatively as a separating technique

2. Gel Electrophoresis
• The pH and other buffer conditions are arranged
so that the molecules being separated carry a net
(negative) charge so that they will me moved by
the electric field toward the positive pole.
• As they move through the gel, the larger
molecules will be held up as they try to pass
through the pores of the gel, while the smaller
molecules will be impeded less and move faster.
This results in a separation by size, with the
larger molecules nearer the well and the smaller
molecules farther away.

22. Electrophoresis of DNA
• The Phosphate groups on the backbone of
the DNA molecule readily give up their H+
ions, therefore nucleic acids are negatively
charged in most buffer systems.
• DNA molecules will migrate away from the
negative electrode (cathode), and migrate
towards the positive electrode (anode).
• The higher the voltage, the greater the
force felt by the DNA molecule, and the
faster they will migrate in an electric field.

23. Electrophoresis: General
Principles
• An idealized, simplified situation: an isolated charged
particle in a nonconducting medium.
• The force experienced by a particle in an electrical field is
given by Coulomb’s law,
• F = ZeE (E-electric field: potential per unit length)
• The viscous resistance of the medium to the motion: -fv
(f: the frictional factor)
• The viscous resistance of the medium just balances the
driving force.
fv = F = ZeE

24. Electrophoresis: General Principles
• Electrophoretic mobility U (the ratio of velocity to the
strength of the driving field)
U = v/E = Ze/f
• If the particle happens to be spherical, Stokes’s law applies
U = Ze/6a
• The zonal techniques: In these methods, a thin layer or zone
of the macromolecule solution is electrophoresed through
some kind of matrix.
• The matrix provides stability against convection. In addition,
in many cases the matrix acts as a molecular sieve to aid in
the separation of molecules on the basis of size.

25. Electrophoresis: General Principles
• The kind of supporting matrix used depends on the type of
molecules to be separated and on the desired basis for separation:
charge, molecular weight, or both.
• Almost all electrophoresis of biological macromolecules is at
present carried out on either polyacrylamide or agarose gels

26. Electrophoretic Separation of DNA
• Agarose Gel Electrophoresis
• Acrylamide Gel Electrophoresis (Native versus
Denaturing Conditions)
• Capillary Electrophoresis

27. ELECTROPHORESIS
DNA and RNA are negatively
charged; they RUN TO RED!

28. Gel Matrices Used for
Electrophoresis of DNA
• Agarose Gels have fairly large pore sizes and
are used for separating larger DNA molecules
(Restriction Fragment Length Polymorphism
Analysis)
• Polyacrylamide Gels are used to obtain high
resolution separations for smaller DNA
molecules (STR analysis and DNA sequence
analysis)

29. Introduction to Agarose
Gel Electrophoresis

30. Principles of Gel Electrophoresis
• The gel itself is composed of either agarose or
polyacrylamide.
• Agarose is a polysaccharide extracted from
seaweed.
• Polyacrylamide is a cross-linked polymer of
acrylamide.
– Acrylamide is a potent neurotoxin and should be
handled with care!

31. Gel Electrophoresis Matrices
Agarose
Acrylamide

32. Agarose Gel Electrophoresis
• Yield Gel – Semiquantitative and qualitative
analysis of isolated DNA
• Separation of DNA restricted with Hae III
(RFLP analysis) followed by a Southern Blot
and Hybridization with a labeled probe
• Post Amplification confirmation and
qualitative assessment of PCR product

33. Assessing DNA Quality
Experiment:
 100 ng K562 DNA
 Digest with DNAse
Molecular Weight Ladder
~23Kbp
~ 2kbp
Time
0
15
30
45
seconds
1
2
3
minutes

34. Types Of Nucleic Acid
Electrophoresis
• Agarose gel electrophoresis
– DNA or RNA separation
– TAE or TBE buffers for DNA, MOPS with
formaldehyde for RNA
• Polyacrylamide gel electrophoresis (PAGE)
– Non-denaturing (Special applications in research)
– Denaturing contain 6-7 M Urea (Most common)

35. Agarose Gel Electrophoresis
• Separates fragments based on mass, charge
• Agarose acts as a sieve
• Typically resolve 200 bp-20 kbp
– fragments <200 bp, polyacrylamide gels
– fragments> 20 kbp, pulse field gels
• Include DNA size standards

36. Agarose Gel Electrophoresis
• An electrophoresis chamber and power supply
• Gel casting trays, which are available in a variety of
sizes and composed of UV-transparent plastic.
• Sample combs, around which molten agarose is
poured to form sample wells in the gel.
• Electrophoresis buffer, usually Tris-acetate-EDTA
(TAE) or Tris-borate-EDTA (TBE).

37. Agarose Gel Electrophoresis
• Loading buffer, which contains something dense
(e.g. glycerol) to allow the sample to "fall" into
the sample wells, and one or two tracking dyes,
which migrate in the gel and allow monitoring or
how far electrophoresis has proceeded.
• A fluorescent dye used for staining nucleic
acids, such as Ethidium bromide, Sybr Green, or
Sybr Gold.
• Transilluminator or Fluorescent Gel Scanner for
photodocumentation

38. Migration of DNA Fragments in
Agarose
• Fragments of linear DNA migrate through
agarose gels with a mobility that is
inversely proportional to the log10 of their
molecular weight

39. Agarose Concentration
• By using gels with different concentrations of
agarose, one can resolve different sizes of DNA
fragments. Higher concentrations of agarose
facilite separation of small DNAs, while low
agarose concentrations allow resolution of
larger DNAs.

40. Agarose Concentration

41. Agarose (%)
Range of separation of linear DNA
(in kilobases)
0.3 60 - 5
0.6 20 - 1
0.7 10 - 0.8
0.9 7 - 0.5
1.2 6 - 0.4
1.5 4 - 0.2
2.0 3 - 0.1

42. Agarose Electrophoresis
Voltage
• As the voltage applied to a gel is increased,
larger fragments migrate proportionally faster
that small fragments. For that reason, the best
resolution of fragments larger than about 2 kb
is attained by applying no more than 5 volts
per cm to the gel (the cm value is the distance
between the two electrodes, not the length of
the gel).

43. Electrophoresis Buffer
• Several different buffers have been recommended
for electrophoresis of DNA. The most commonly
used for duplex DNA are TAE (Tris-acetate-EDTA)
and TBE (Tris-borate-EDTA). DNA fragments will
migrate at somewhat different rates in these two
buffers due to differences in ionic strength.
Buffers not only establish a pH, but provide ions
to support conductivity. If you mistakenly use
water instead of buffer, there will be essentially
no migration of DNA in the gel! Similarly, if you
use concentrated buffer (e.g. a 10X stock
solution), enough heat may be generated in the
gel to melt it.

44. Gel Electrophoresis: Apparatus and Types of
Gels
• Horizontal Gel Units (“Submarine Gels”)
– Most DNA and RNA gels
– Agarose
• Vertical Gel Units
– Polyacrylamide gels
– Typically sequencing gels
• Pulse Field Gel Units
– Any electrophoresis process that uses more than one
alternating electric field
– Agarose
– Large genomic DNA (Chromosomal)

45. Electrophoresis Equipment: Horizontal or
Submarine Gel
DNA/RNA is negatively charged: RUN TO RED

46. Agarose Gel Electrophoresis System