VARIABLE CONCENTRATION OF AGAROSE GELS AND DISTINCT APPLICABILITY CRITERIA

Zachariou Angelina¹, Pandis Spyridon¹, Velegraki Aristea² and Kambouris Manousos E.¹

¹Dept of Medical Laboratories, Fcty of Health and Caring Professions, Technological Educational Institute of Athens, Ag Spyridonos st., 12210 Athens, GREECE.

² Mycology Laboratory, Medical School, National & Kapodistrian University of Athens

75-77 Mikras Asias st., 11527 Athens, GREECE

Abstract

The purpose of this experiment was to study the discriminatory capacity and other functional parameters of varying agarose gel concentrations, and to create a documented classification according to concentration and ability to separate DNA fragments of various sizes with analytical efficiency.

The experiment utilized agarose-TBE gels at concentrations of 0.5%, 0.8%, 1.2%, 1.6%, 2%, 2.5%, 3% and 3.5%. Electrophoresis was performed using randomly selected amplicons from fungal PCR reactions and a molecular marker (100-bp DNA ladder). Each electrophoresis was normalized to the migrating first loading buffer dye, bromophenol blue, reaching the bottom of the gel.

The gels were photographed and widely available computer software tools (Microsoft Excel, Microsoft Word) were utilized to compile mobility - band size standard curves for each gel and resulted in charts indicating the alteration of electrophoretic properties in correlation with concentration. Specifically, the co-migration of DNA ladder bands and the loading buffer dyes was examined, and the Discriminatory Power, the Resolution Spectrum, and other correlated measurements and parameters were defined, calculated for each gel, and plotted for the whole gel range.

Based on the results, we suggest the use of varying gel concentrations, according to the application sought and the prospective, expected or known fragment size to undergo electrophoresis.

Keywords: agarose gel electrophoresis, standard curve, DNA mobility, discriminatory power

[return]