Simple ethidium bromide question
Bryan L. Ford
fordb at bcc.orst.edu
Wed Aug 27 21:04:42 EST 1997
Hiranya Roychowdhury wrote:
> At 03:57 PM 8/26/97 GMT, Pascal_Bochet wrote:
> >I was wondering why is it that in PCR when you look at the products on a
> >agarose gel stained with ethidium bromide, you can see primer dimer if
> there is
> >any but you cannot see single stranded primer (if for instance you omit TAQ or
> >dNTP from the reaction.) Is it the size or the fact that it is single stranded
> >? In other terms what does ethidium bromide exactly bind to?
> >Thank you for any insight.
> >Pascal_Bochet at brown.edu
> EtBr intercalates into the "grooves" of double-stranded nucleic acids. This
> is the reason why the rRNA molecules, which have extensive secondary
> structures, light up brighter on the gels. Short NA's do not provide such
> secondary structures to allow EtBr intercalation.
Recently I have done extensive electrophoresis on 20% polyacrylamide
of single stranded oligonucleotides of 17 through 20 bases length. I
found, to my surprise, that ethidium bromide staining strongly
visualized 50 pmole per lane loadings, and appeared to give adequate
fluorescence with 10 pmole per lane or less.
I believe that the loss of signal often seen with the smaller oligos
(e.g. "primer dimers") can be attributed in large part to the loss of
these short oligos to migration and elution during staining and
destaining in lower percentage gels. With the 20% gels the staining and
destaining times become relatively unimportant.
I have no reason to question the established view that EtBr is an
intercalator, but one has to wonder just how it is able to give good
staining of such short single stranded pieces. Perhaps with relatively
high concentrations of oligos we have sufficient strand-strand
hybridization to allow intercalation.
Bryan L. Ford
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