Commercial automated gel electrophoresis apparatus: application to DNA, band dispersion, nonlinear Ferguson curves, and isolation.
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 micrograms 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Chrambach
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A
Section on Macromolecular Analysis, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-0001,USA.
Yarmola
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E
Zakharov
S F
SF
Garner
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MM
D002021:Buffers; D004247:DNA / Q000032:analysis*; D004587:Electrophoresis, Agar Gel / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D012984:Software; D013997:Time Factors
D002021:Buffers; D004247:DNA