The Agarose Matrix
A natural colloid extracted from seaweed, agarose is a linear polysaccharide made up of the repeating unit agarobiose, which consists of alternating units of 1,3-linked b-D-galactopyranose and 1,4-linked 3,6-anhydro-a-L-galactopyranose. Gels prepared from agarose have a substantially larger pore size than polyacrylamide gels. Agarose gels can be prepared faster than polyacrylamide, but their resolution is inferior, especially for smaller molecules. For this reason, agarose gels have limited application for the electrophoresis of small nucleic acid fragments and most proteins. However, for the electrophoretic separation of larger nucleic acid molecules (>400 bp), agarose presents an excellent choice. It is easier to handle and safer to prepare than polyacrylamide.
A potentially significant problem in agarose electrophoresis is electroendosmosis (EEO). Electroendosmosis in electrophoresis refers to the flow of water under the influence of an electric field due to immobilized charge groups on the matrix, primarily sulfate and carboxyl groups. While the sulfate and carboxyl groups cannot migrate, their counter ions do migrate, moving toward the cathode, causing a net flow of water through osmosis in that direction. In the past, this problem often led to badly smeared bands. Fortunately, purification methods have been developed which nearly eliminate this problem in modern agarose electrophoresis. The use of a high-quality electrophoresis grade agarose, such as one of National Diagnostics' AquaPor agaroses, will prevent electroendosmosis effects.
|AquaPor LE||AquaPor LM||AquaPor HR||AquaPor ES|
|EEO (-m)||= 0.12||= 0.05||= 0.12||= 0.05|
|=1200 @ 1.0||= 450 @ 1.0%
= 1700 @ 3.0%
|= 400 @1.5%
= 1100 @ 3.0%
|= 1700 @ 1.0%
= 3200 @ 1.5%
|= 37 @ 1.0%||= 27 @ 1.5%||= 26 @ 1.5%
= 35 @ 3.0%
|= 40 @ 1.5%|
|= 92 @ 1.0%||= 65 @ 1.5%||= 66 @ 1.5%
= 75 @ 3.0%
|= 66 @ 1.5%|
|= 0.20||= 0.20||= 0.20||= 0.20|
AquaPor LE is general-purpose agarose often used for Northern and Southern blotting and restriction mapping. AquaPor LM is a low melting point agarose used for sample recovery, probe purification, and in-gel PCR and ligation. AquaPor HR is high resolving agarose that is also low melting used for PCR analysis, probe purification, and in-gel enzyme reactions. AquaPor ES is high-strength agarose used for pulsed-field gel electrophoresis (PFGE) and field inversion gel electrophoresis (FIGE).
NEXT TOPIC: Electrophoresis Buffers