sizing ssDNA

Paul N Hengen pnh at fcsparc6.ncifcrf.gov
Tue Jul 27 17:00:34 EST 1993


In article <CAFL9A.GHK at news.Hawaii.Edu> marilou at ahi.pbrc.hawaii.edu
 (Marilou Andres) writes:

> I have prepared ssDNA from pBS KS+ plasmid containing the f1 replication site
> by infecting with the R408 phage. One of the ways some people suggested that I
> can be sure of what I have is indeed ssDNA is to run my sample along with single
> stranded molecular weight marker in the gel.  However, such a product does not
> seem to exist in the market.  Has anyone out there come across a single
> stranded molecular weight market?  If not, is there any other way of
> determining whether a DNA sample is single stranded?

This is from the Bulletin (supplement 15; summer 1991) that accompanies Ausubel,
F. M., R. Brent, R. E.  Kingston, D. D. Moore, J. G.  Seidman, J. A. Smith, and
K. Struhl. 1991.  Current Protocols in Molecular Biology. John Wiley and Sons,
New York:

  When using phagemids I often find that some single-stranded (ss) DNA preps
  have become contaminated with double-stranded (ds) DNA.  Because of the
  anomolous migration of ss DNA through agarose gels, the identification of ss
  DNA by molecular weight is not practical.  Although ds DNA does not
  necessarily interfere with subsequent ss techniques (such as sequencing or
  mutagenesis) it is advantageous to know the quality of a ss prep and the
  amount of ds contamination.  Staining an agarose gel with acridine orange and
  viewing under UV light enables one to distinguish ss DNA (which fluoresces
  red) from ds DNA (fluoresces green).  Simply stain the gel with 300 ug/ml
  acridine orange in 10 mM NaPO4, pH 7.3 (0r in H2O), and destain in the same
  solution (NaPO4 or H2O).  Reference:  McMaster and Carmichael, 1977, Analysis
  of single- and double-stranded nucleic acids on polyacrylamide and agarose
  gels by using glyoxal and acridine orange, Proc. Nat. Acad. Sci. 74:4835-4838.

  - Karen Martell, University of Michigan, Ann Arbor

Paul N. Hengen
National Cancer Institute
Frederick Cancer Research and Development Center
Frederick, Maryland 21702-1201 USA



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