title: Modification of gel architecture and TBE/TAE buffer composition to minimize heating during agarose gel electrophoresis.
abstract: Agarose gel electrophoresis of DNA and RNA is routinely performed using buffers containing either Tris, acetate, and EDTA (TAE) or Tris, borate, and EDTA (TBE). Gels are run at a low, constant voltage (∼10 V/cm) to minimize current and asymmetric heating effects, which can induce band artifacts and poor resolution. In this study, alterations of gel structure and conductive media composition were analyzed to identify factors causing higher electrical currents during horizontal slab gel electrophoresis. Current was reduced when thinner gels and smaller chamber buffer volumes were used, but was not influenced by agarose concentration or the presence of ethidium bromide. Current was strongly dependent on the amount and type of EDTA used and on the concentrations of the major acid-base components of each buffer. Interestingly, resolution and the mobilities of circular versus linear plasmid DNAs were also affected by the chemical form and amount of EDTA. With appropriate modifications to gel structure and buffer constituents, electrophoresis could be performed at high voltages (20-25 V/cm), reducing run times by up to 3-fold. The most striking improvements were observed with small DNAs and RNAs (10-100 bp): high voltages and short run times produced sharper bands and higher resolution.
authors.affiliation: Chemistry and Biochemistry, Texas State University, 601 University Drive, San Marcos, TX 78666, USA.
authors.forename: Brian A
authors.initials: BA
authors.lastname: Sanderson
authors.affiliation: Chemistry and Biochemistry, Texas State University, 601 University Drive, San Marcos, TX 78666, USA.
authors.forename: Naoko
authors.initials: N
authors.lastname: Araki
authors.affiliation: Chemistry and Biochemistry, Texas State University, 601 University Drive, San Marcos, TX 78666, USA.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Lilley
authors.affiliation: Chemistry and Biochemistry, Texas State University, 601 University Drive, San Marcos, TX 78666, USA.
authors.forename: Gilberto
authors.initials: G
authors.lastname: Guerrero
authors.affiliation: Chemistry and Biochemistry, Texas State University, 601 University Drive, San Marcos, TX 78666, USA. Electronic address: LL18@txstate.edu.
authors.forename: L Kevin
authors.initials: LK
authors.lastname: Lewis
mesh_terms: D000085:Acetates / Q000737:chemistry*; D001888:Boric Acids / Q000737:chemistry*; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification; D004492:Edetic Acid / Q000737:chemistry*; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D005029:Ethylenediamines / Q000737:chemistry*; D005782:Gels; D006358:Hot Temperature; D013997:Time Factors; D014325:Tromethamine / Q000737:chemistry*
chemical_list: D000085:Acetates; D001888:Boric Acids; D005029:Ethylenediamines; D005782:Gels; C069591:Tris-borate-EDTA buffer; C115179:tris-acetate-EDTA buffer; D014325:Tromethamine; D004247:DNA; D004492:Edetic Acid
keywords: Agarose; Current; Gel electrophoresis; Nucleic acids; Resolution