Gel Electrophoresis

The term electrophoresis means any experimental technique that is based on movement
of charged particles (ions, molecules, macromolecules) in electric field in liquid medium.
Any electrically charged particle dissolved in aqueous solution, when placed to a constant
electric field, will start to migrate towards the electrode bearing the opposite charge; the speed of
the particle movement will be directly proportional to the applied voltage and particle charge, but
inversely proportional to the particle size. Any molecules that differ in size and/or charge can be
separated from each other in this way. The electrophoretic analysis can in principle be applied to
any particles that are charged under given experimental condition, such as small cations or
anions, organic acids, amino acids, peptides, saccharides, lipids, proteins, nucleotides, nucleic
acids, even the whole subcellular particles or the whole cells. In practice, however, the by far
commonest subjects of electrophoretic separation are proteins and nucleic acids.

Gel

Electrophoresis is usually performed in some support, which can be paper, cellulose

acetate, or gel made of starch, agarose or polyacrylamide. The latter two materials are used by
far most often. The support – gel – can be envisaged as a three dimensional structure of open
pores filled with a liquid; this space structure prevents convection flows of electrophoretic buffer
during separation. Simultaneously, however, size of the gel pores, which especially in
polyacrylamide can be easily controlled, represents another important factor affecting result of an
electrophoretic separation. If the pores are large enough to allow free movement of
macromolecules, the separation of Molecules will be the same as in free solution, i.e. according
to the charge/size ratio (more exactly stated: according to the surface charge density). On the
other hand, a thicker gel with small pores will present a mechanical obstacle to the movement of
molecules, i.e., the gel will act as a ‘molecular sieve’, slowing down the protein movement the
more the bigger the molecules are. Nucleic acids, whose charge density does not depend on their
size (every other nucleotide bears the same charge), can actually be separated only according to
their size, i.e., in gels dense enough to act as molecular sieves.
Recipe for Agarose Gel Electrophoresis

The equipment and supplies necessary for conducting agarose gel electrophoresis are
relatively simple and include:

 An electrophoresis chamber and powersupply

 Gel casting trays, which are available in a variety of sizes and composed of UV-
transparent plastic. The open ends of the trays are closed with tape while the gel is
being cast, then removed prior to electrophoresis.
 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).
 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 visual monitoring or how far the electrophoresis has proceeded.
 Ethidium bromide, a fluorescent dye used for staining nucleic acids. NOTE: Ethidium
bromide is a known mutagen and should be handled as a hazardous chemical - wear
gloves while handling.
 Transilluminator (an ultraviolet lightbox), which is used to visualize ethidium bromide-
stained DNA in gels. NOTE: always wear protective eyewear when observing DNA on a
transilluminator to prevent damage to the eyes from UV light.

PROCEDURE
1. Preparation of Gel
Agarose gel is prepared by weight volume %. For 0.8 % used for DNA sample take 0.8
grams of Agarose and add it to TAE solution to make total volume 100 ml. For PCR sample
usually 2% gel is prepared using 2 gram agarose and dissolving it in TAE buffer.
2. Polymerization of Gel
Gel is poured into gel cassette and comb is placed for wells, then it is allowed to cool for
almost 30 min to polymerize
3. Electrophoresis Buffer
Electrophoresis buffer is poured into tank and gel is placed in it and comb is removed
4. Loading of Samples
Samples are loaded into wells along with dye. DNA marker is also loaded. Markers
contains fragments of known base pairs. This helps in identification of required DNA
fragments
5. Voltage
The best resolution of fragments larger 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).
6. Staining and Visualization
When adequate migration has occurred, the gel is placed on an ultraviolet
transilluminator at wavelength of 254nm. DNA fragments are visualized by staining with
ethidium bromide and placing it in UV light. This fluorescent dye intercalates between
bases of DNA and RNA. It is often incorporated into the gel so that staining occurs during
electrophoresis (Pre Staining), but the gel can also be stained after electrophoresis by soaking
in a dilute solution of ethidium bromide (Post Staining).

PRECAUTIONS
1. While loading samples into well try not to push hard, it will destroy wells
2. Ethidium bromide is carcinogenic , it should be handled with great care
3. Voltage should not be high
4. Gel should be made in same TAE buffer that is used as running buffer
Gel electrophoresis based image analysis. Agarose gels, stained by Ethidium bromide
and UV light