Agarose
Agarose is a natural linear polysaccharide that comes from seaweed (red algae) and consists of the D-galactose and 3,6-anhydro-L-galactose repeating units. It forms an inert matrix used in electrophoresis, chromatography and other molecular biology and biochemistry techniques. The molecular composition of agarose, mostly looks like a porous substance, mesh or net which depends on the amount of agarose form a web of channels with diameters from 50 nm to 200 nm. Its structure provides the ability to slow down the movement of DNA and other molecules in a way that smaller molecules travel quicker through the agarose gel, while, bigger molecules moves slower, this ability leads to separation of DNA fragments in a process called gel electrophoresis.
Lack of toxicity makes working with agarose very convenient and in combination with its other properties and specifications make it useful as a gelling agent in many biological and biochemical applications, such as nucleic acid electrophoresis, gel chromatography, affinity chromatography, ion exchange chromatography, immunodiffusion techniques, gel plates or overlays for cells in tissue culture and cell culture media.
Key agarose properties
Sulfate content: Since sulfate is the major ionic group present in Agarose, its content is considered as an indicator of purity.
Gel strength: The force that must be applied to a defined area of the gel to cause it to fracture.
Gelling point (Temperature): The temperature at which an aqueous agarose solution forms a gel as it cools. Agarose solutions shows hysteresis in the liquid-to-gel transition; hence, their gel point and the melting point are not the same.
Melting point (Temperature): The temperature at which agarose forms an aqueous solution as it warms.
Electroendosmosis (EEO): The movement of liquid in a porous material due to an applied electric field is called Electroendosmosis (EEO). Anionic groups in an agarose gel cannot move as they are affixed to the matrix; dissociable counter cations however, are able to migrate toward the cathode which rising the EEO. Since biopolymers are usually moving electrophoretically toward the anode, EEO can disrupt separations because of internal convection. Therefore, the low EEO leads to better movement and separation.
Based on these properties the agaroses are classified to different categories
Agarose LE (Low electroendosmosis)
is the most abundantly used agarose for DNA gel electrophoresis, whereby, low electroendosmosis (EEO), describes the electrically influenced movement of material through gel. Agarose LE increases mobility, and reduces band distortion caused by counterflow. It promotes increased electrophoretic mobility, results in improved resolution and shorter run times. Low EEO Agarose also allows migration of larger particles such as viruses through the matrix.
Agarose LM (Low Melting temperature agarose)
is a chemically modified agarose whereby, hydroxyethyl groups have been introduced into the agarose molecule. This chemical modification decreases the gelling temperature to ˜ 26 °C and the melting temperature at 65 °C. Low melting temperature allows for the recovery of undamaged nucleic acids below denaturation temperature. Easily digested by agarose enzymes, makes Agarose LM an ideal option to recover large DNA fragments suitable for cloning or enzymatic processing. Agarose LM has low EEO (electroendosmosis, ≤0.12), allowing for sharp DNA banding and high resolution.
Agarose HR (High Resolution)
is a high quality, Molecular Biology Grade Agarose, suitable for the separation of small DNA fragments and PCR products between 20 – 1000 bp. Due to its improved resolution, it is suitable for applications that require efficient separation of small DNA fragments and PCR products. Agarose HR with an excellent clarity of gels at a concentration of as high as 5%, allows the separation of DNA fragments differing by 2% in molecular weight and provides a good alternative to polyacrylamide electrophoresis. The gelling and melting temperature of this agarose is lower than Agarose LE and higher than Agarose LM.
Pulsed Field Gel Electrophoresis (PFGE) Agarose
is a high gel strength agarose, designed to separate large DNA fragments at lower concentrations. Small DNA molecules (less than 20 kb) can be separated by standard gel electrophoresis technique as they move in a size-dependent manner, however, DNA larger than 20-25 kb move together through the gel in a size-independent manner and appear in a gel as a single large diffuse band, and therefore, the standard gel electrophoresis technique is unable to separate very large DNA molecules effectively.
Pulsed Field Gel Electrophoresis (PFGE) is a technique used to separate large DNA molecules by applying an electric field to a gel that periodically alters direction. The various size of DNA molecules react differently to periodic altering of field direction, whereby, larger DNA fragments will be slower to realign their charge in response to alteration of field direction, while smaller pieces will react quicker. Over the course of time with the consistent altering of field directions, each DNA fragment will begin to separate more and more even at very large lengths.
Appropriate percentage of agarose gel for DNA electrophoresis
The percentage of agarose gel used for DNA electrophoresis depends on the size of DNA. The larger DNA fragment you are working with; the lower concentrated agarose you need.
|
Agarose Concentration (%) |
DNA Size (bp) |
|
0.5 |
1,000 – 30,000 |
|
0.75 |
750 – 12,500 |
|
1.0 |
500 – 10,000 |
|
1.2 |
300 – 7,500 |
|
1.5 |
150 – 3,000 |
|
2.0 |
50 – 1,500 |
|
3.0 |
25 – 1,000 |
|
4.0 |
10 – 500 |
|
5.0 |
10 – 300 |
Appropriate volume of agarose gel for different gel tray
The volume of agarose gel needed depends on the size of gel casting tray. The bigger tray, the higher volume. Here is some suggestion based on gel tray dimensions.
|
Gel Tray Dimensions (cm) |
Agarose Gel Volume Range |
|
7 x 8 cm |
40 ml |
|
9 x 11 cm |
70 – 80 ml |
|
12 x 14 cm |
120 – 130 ml |