Electrophoresis technique used for separation of Macromolecules(DNA, Proteins & their derivatives) Separation occur on the basis of charge to size ratio

1. Practical #6 14-nov-2018
Running of Agarose GelElectropherosis
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.

2. Recipe for Agarose Gel Electrophoresis
The equipment and supplies necessary for conducting agarose gel electrophoresis are
relatively simple and include:
 An electrophoresis chamber and power supply
 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

3. 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 occured, the gel is placed on a 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

4. Gel electrophoresis based image analysis. Agarose gels, stained by Ethidium bromide
and UV light