Discontinuous buffer system for polyacrylamide and agarose gel electrophoresis of DNA fragments.
DNA fragments up to 9 kb in size were stacked and separated by polyacrylamide gel electrophoresis, and those up to 50 kb in size by agarose gel electrophoresis, using a discontinuous buffer system. Polyacrylamide gels at pH 8.9, 2 degrees C, 0.01 M ionic strength, yielded sharp bands with DNA loads of 8 micrograms/cm2 of gel of a mixture of 19 DNA fragments in the size range of 72-23130 bp, while agarose gels at pH 8.5, 25 degrees C, provided well-resolved, unperturbed bands at 0.04 M ionic strength with DNA loads of 1 microgram/cm2 of the same mixture. Note that the ionic strength of the agarose gels is comparable to the conventionally used 0.5 x TBE (Tris-borate-EDTA) buffer, while that successfully applied to polyacrylamide is seven-fold less than the ionic strength of conventionally used 1 x TBE buffer, with a substantially shorter duration of electrophoresis as a result. The application of a discontinuous buffer system to the gel electrophoresis of DNA results in (i) Band identification by Rf, the migration distance relative to a sharply defined "buffer front" (moving boundary). This is sufficiently labor saving, compared to determining absolute mobilities, so as to render practical the expression of bands as numbers, with benefits for data storage, statistical manipulations and physico-chemical exploitation of mobility data. The use of Rf's also circumvents loss of precision in mobility measurement resulting from progressive band spreading of dye bands used as a front. (ii) A uniformly and highly concentrated starting zone, beneficial to resolution, is obtained, without the losses by which separate concentration steps are usually burdened.(ABSTRACT TRUNCATED AT 250 WORDS)
Orbán
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L
Section on Macromolecular Analysis, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892.
Chrambach
A
A
D001482:Base Composition; D002021:Buffers; D003432:Cross-Linking Reagents; D004247:DNA / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D009994:Osmolar Concentration
D002021:Buffers; D003432:Cross-Linking Reagents; D004247:DNA