ELECTROPHORESIS
OF DNA FRAGMENTS IN AGAROSE GELS

General Consideration

Any charged ion or group at any pH other than their isoelectric point, in an electric field, migrate at a rate proportional to:
1. Their charge density.
2. Their weight, the size and shape of the molecule.
3. The environmental conditions (ionic strength, viscosity, temperature and the kind of support matrix).

Electrophoresis is a technique used to separate and sometimes purify macromolecules - especially proteins and nucleic acids - that differ in size, charge or conformation.

When charged molecules are placed in an electric field, they migrate toward either the positive (anode) or negative (cathode) pole according to their charge density, their weight, size and shape of the molecule, and the environmental conditions (ionic strength, viscosity, temperature and the kind of support matrix).
Some Properties of Nucleic acids:
DNA molecules have much higher molecular weights than proteins.
The phosphate backbone of DNA is highly negatively charged.
The Nucleic acids are polyanionic molecules uniformly charged and therefore the molecules will be separated according to their molecular weight.
Nucleic acids come in a very wide range of sizes, from several dozen base pairs to many millions.

Support Matrix:
The most used material as matrix is agarose or polyacrylamide gel. Agarose, because of its large range of pore size, is mostly used to separate micro and macromolecules ( such as nucleic acids, large proteins, protein complexes).

Polyacrylamide is a cross-linked polymer of acrylamide. Generally it makes smaller pore gels, and therefore is suitable for the separation of most proteins and smaller nucleic acid fragments, but it is toxic. The length of the polymer chains is dictated by the concentration of acrylamide used, which is typically between 3.5 and 20%. Polyacrylamide gels are significantly more annoying to prepare than agarose gels.
Because oxygen inhibits the polymerization process, they must be poured between glass plates.
Acrylamide is a potent neurotoxin and should be handled with care! Wear disposable gloves when handling solutions of acryl amide, and a mask when weighing out powder. Polyacrylamide is considered to be non-toxic, but Polyacrylamide gels should also be handled with gloves due to the possible presence of free acrylamide.

Polyacrylamide gels have a rather small range of separation, but very high resolving power. In the case of DNA polyacrylamide is used for separating fragments of less than about 500 bp. However, under appropriate conditions, fragments of DNA differing is length by a single base pair are easily resolved. In contrast to agarose, polyacrylamide gels are used extensively for separating and characterizing mixtures of proteins.
Agarose:
Agarose is a polysaccharide. It is typically used at concentrations of 0.5 to 2%. The higher the agarose concentration the "stiffer" the gel. Agarose gels are extremely easy to prepare: you simply mix agarose powder with an appropriate buffer solution, melt it by heating, and pour the gel. It is non-toxic.
Agarose gels have a large range of separation, but relatively low resolving power. By varying the concentration of agarose, fragments of DNA from about 100 to 30,000 bp can be separated using standard electrophoretic techniques.
The gels are run horizontally in an adequate buffer usually at room temperature.

Agarose Gel electrophoresis is the standard technique to separate, identifies, and purifies DNA fragments according to their size.
Also, this method is employed to check the progression of a restriction enzyme digestion, to quickly determine the yield and purity of a DNA isolation or PCR reaction, and to size fractionated DNA molecules.
The technique is simple, rapid to perform, and capable of resolving mixtures of DNA fragment that can not be separated adequately by other sizing procedures.

The percentage of agarose in the gel varies, and affects the migration of the fragments. A DNA fragment of given size migrates at different rates through the gels containing different concentrations of agarose.
A 0.7% agarose gel shows good separation of large DNA fragments (5-10kb) and a 2% gel will show good separation for small fragments (0.2-1kb). For separation of smaller fragments higher concentration can be used.
Closed circular, nicked circular and linear DNA of the same MW migrates through the agarose gels at different rates. The relative mobilities of these three DNA forms are dependent primarily on the agarose concentration in the gel but are also influenced by the strength of the applied current, the ionic strength of the buffer, and the density of super helical twists in the DNA.
The electrophoretic behavior of DNA in agarose gels (by contrast to polyacrylamide gels) is not significantly affected either by the base composition of the DNA or the temperature at which the gel is run.
The phosphate molecules that make up the backbone of DNA molecules have a high negative charge. When DNA is placed on an electrical field these negatively charged DNA molecules migrate toward the positive end of the field.

Hellabio Precast Agarose Gels ( HPAG) are ready to use and can be offered in four size of gels.
According to the User’s demands it is possible to offer precast agarose gels to fulfil any needs (gel size, number and capacity of wells, size range of fragments) and to get easily the best results at a very moderate cost.
The User of the HPAG is insured to get rapid and reproducible results and to save time and money.

Hellabio offers a complete line of nucleic acid electrophoresis products such as a broad range of molecular biology grade pre-cast agarose gels, electrophoresis tank and all necessary accessories, so that the user works independent and get comfortable the best resolutions result.

Hellabio Electrophoresis Systems [Submarine and Vertical] are offered for the standard size gel range. In the same time the user has the ability to choose a variety of gel lengths and comb sizes according to his protocol

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