Discontinuous buffer system for polyacrylamide and agarose gel electrophoresis of DNA fragments
Orbán, L. and Chrambach, A. (1991) Discontinuous buffer system for polyacrylamide and agarose gel electrophoresis of DNA fragments. Electrophoresis, 12 (4). pp. 233-240.
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DNA fragments up to 9 kb in size were stacked and separated by polyacrylamid gel electrophoresis, and those up to 50 kb in size by agarose gel electrophoresis using a discontinuous buffer system. Polyacrylamide gel at pH 8.9, 2°C, 0.01 M ionic strength, yielded sharp bands with DNA loads of 8 μg/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°C, provided well-resolved, unperturbed bands at 0.04 M ionic strength with DNA loads of 1 μg/cm2 of the same mixture. Note that the ionic strength of the agarose gels is comparable to the conventionally used 0.5 × TBE (Tris-borate-EDTA) buffer, while that successfully applied to polyacrylamide is seven-fold less than the ionic strength of conventionally used 1 × TBE buffer, with is 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 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. (iii) The degree of dilution of the DNA sample becomes unimportant.
|Publication Type:||Journal Article|
|Publisher:||John Wiley & Sons Ltd|
|Copyright:||© 1991 VCH Verlagsgesellschaft mbH|
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