ISSN:
0173-0835
Keywords:
Gel electrophoresis
;
Automation
;
Dispersion
;
Preparativeness
;
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
Recently available commercial automated gel electrophoresis apparatus with intermittent scanning of fluorescently labeled gel patterns (the HPGE-1000 apparatus of LabIntelligence, Menlo Park CA) was tested with regard to (i) its applicability to DNA in its native conformation, (ii) its ability to recognize the correct number of components, (iii) its capability to evaluate the width and shape of bands detected during electrophoresis, (iv) its ability to yield nonlinear Ferguson plots in a labor-saving fashion, and (v) its preparative potential. Ethidium homodimer (EtD) DNA (bp) ratios were systematically varied and the mobility of DNA fragments labeled at each ratio was measured in order to find a ratio which provided an unaltered mobility and presumably therefore an unaltered conformation of the fragment. That ratio was found to be 1/40 EtD/ DNA (bp) or less. With such weak labeling of DNA, a representative fragment of 527 bp length requires a minimum load of 200 ng and a 2 μg load for a full-scale peak height. Using the baseline automatically selected by the software of the apparatus, the band areas of the 17 components of a DNA digest were consistently evaluated by the software, as evidenced by the proportionality between DNA length and area. The areas of the separated bands of DNA fragments of 1857 and 121 bp length were found to be constant with time of electrophoresis. The dispersion coefficient was found to decrease with agarose concentration in electrophoresis at 1 V/cm; however, at higher field strength, the band width of the 1857 bp fragment was surprisingly found to increase with gel concentration, presumably due to stretching. Electrophoretic band dispersion was not found to be due to diffusion since zone spreading in the absence of the electric field under otherwise identical conditions does not proceed measurably. For the discontinuous buffer system of Wiltfang et al. (Electrophoresis 1993, 12, 352-366) the resolving power, in terms of theoretical plate equivalents, was approximately threefold higher compared to that in the Trisborate-EDTA (TBE) buffer under otherwise identical conditions. Two electrophoretic runs of the apparatus with different gel concentrations in its eight channels suffice to define nonlinear Ferguson curves, provided that the free mobility is measured by capillary electrophoresis. The commercial automated apparatus excels as a preparative device by allowing one to monitor the qualitative and quantitative success of electroelution.
Additional Material:
3 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/elps.11501601115
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