Improved stability and electrophoretic properties of preformed fluorescent cationic dye-DNA complexes in a taps-tetrapentylammonium buffer in agarose slab gels.
High-resolution capillary electrophoresis sizing of preformed complexes of bis-intercalating fluorescent dyes with double-stranded DNA has been demonstrated using hydroxyethylcellulose and 3-[tris-(hydroxymethyl) methylamino]-1-propanesulfonic acid-tetrapentylammonium (Taps-NPe+4) buffers (S. M. Clark and R. A. Mathies, Anal. Chem. 69, 1355-1363, 1997). Such capillary electrophoresis separations were unattainable in conventional buffers containing other cations such as Tris+, Na+, and NH+4. We report here the behavior of preformed double-stranded DNA-dye complexes on agarose slab gel electrophoresis in 40 mM Taps-NPe+4, 1 mM H2EDTA, pH 8.2. Upon electrophoresis in this buffer (a) complexes formed at DNA base pairs:dye ratios ranging from 100:1 to 5:1 show the same mobility; (b) the half-lives of DNA-dye complexes with monointercalators are two- to threefold longer than those in commonly used Tris buffers; (c) there is little dye transfer between labeled and unlabeled DNA molecules; and (d) precise two-color sizing of preformed restriction fragment-dye complexes with fluorescent bisintercalators is achieved.
Zeng
Z
Z
Department of Molecular and Cell Biology, University of California, Berkeley 94720, USA.
Clark
S M
SM
Mathies
R A
RA
Glazer
A N
AN
D002021:Buffers; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D013451:Sulfonic Acids / Q000737:chemistry
D002021:Buffers; D005456:Fluorescent Dyes; D000644:Quaternary Ammonium Compounds; D013451:Sulfonic Acids; C108789:3-(tris(hydroxymethyl)methylamino)-1-propanesulfonic acid; D004247:DNA; C027435:tetrapentylammonium