Low Fluorescence DNA Gels

Bryan L. Ford fordb at bcc.orst.edu
Wed Aug 13 21:02:21 EST 1997

WSchick at aol.com wrote:
> Subject: low fluorescence gels
> We are debating the ultimate sensitivity that one can detect DNA by EtBr or
> SYBRgreen staining, and think the limitation is from the gel background or
> absorption rather than the detection method.
> The AlphaInnotech ChemiImager, by cooling a CCD chip to -40 deg C. in the
> camera, can detect about 200 to 500 fold lower concentration of  DNA on
> chemiluminescent blots than an uncooled camera.  But the fluorescence
> detection from DNA agarose gels is only marginally improved with cooling.  Is
> the gel itself limiting the detection?
> What other media can we use that would have lower UV interference than
> agarose?  Or are there purer agaroses that would permit lower than 0.1ng DNA
> detection?  Could the gel be treated in some way?
> Thanks for your suggestions.

Just a few comments on this topic.

Sometime ago I experimented with "pushing the envelope" of EtBr
fluorescent sensitivity. I found that there are some fundamental factors
that limit EtBr and SYBR. One of these is the presence of a deep red
band from the UV lamps used in most transilluminators (and presumably
epi-illuminators). The source of this red emission may well be a
spectroscopic line from the Hg arc at about 691 nm, but also there may
be some contribution from phosphors in the 300 and 350 nm lamps. So with
the standard orange filter in place, you will have this "background" for
any long exposures. Perhaps you have noticed that long exposures on
transilluminators will eventually show the flourescent bulbs themselves
(>than about 3 s at f5.6, ISO 3000 on ours). The available black light
filters forming the glass surface of transilluminators appear to pass at
least some of this red line. Even if you purchase an expensive
interference filter (don't do it!) to block the red line selectively you
will see that the gel has its own characteristic fluorescence. This
could be a function of the buffer ingredients-- so it might be worth
trying TBE and Taurine buffers.

For our electrophoresis work we have favored the use polyacrylamide, it
always appears to give less background fluorescence-- however this
observation may only be due to the fact that the agarose slabs are
always much thicker than acrylamide verticals. Another benefit of
polyacrylamide is that the DNA capacity is much greater per unit
volume-- this could allow one to concentrate the samples by many fold
and load it into very small wells and thus increase the signal to
background ratio.

 But in any case you may be more or less stuck with agarose if your
products are larger than 1.5 kb or so.

Another comment: We have found that for double-stranded DNA the only
significant advantage of SYBR is that it needs no destaining, whereas
good destaining is essential with EtBr. SYBR can be useful for
single-stranded visualization, but silver staining would be far less
expensive (although less convenient) in this application.

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