Low Fluorescence DNA Gels

Frank O. Fackelmayer fof1 at chclu.chemie.uni-konstanz.de
Thu Aug 14 03:29:26 EST 1997


In article <33F2672D.3EAE at bcc.orst.edu>, fordb at bcc.orst.edu wrote:

> 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.
> 
> 
> 
snip
> 
> 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.


Hi Bryan,
I know that most people say that destaining is necessary for EtBr
detection of DNA in agarose gels. We NEVER destain! It is absolutely not
necessary, and we consider it wasted time. The problem might be that most
people use WAY to much EtBr, so they have to get rid of the excess
afterwards. Thats how we do it:

*run gel IN ABSENCE of EtBr; we use TAE buffer
*stain for 15min at RT, with EtBr diluted 1:10.000 from a 1% stock
solution. We dilute in water, but if it could be necessary to return your
gel to the tank and let it run longer, dilute in electrophoresis buffer.
You will not see the orange color of EtBr in this dilution, but still its
more than enough to give you a sensitivity down to 5-10ng of DNA (on a
6x8cm minigel). Also, the staining solution can be re-used at least
10times, without affecting the sensitivity. For storage, keep it in a
brown glass. 
*after 15min, rinse briefly with water (10sec is enough) end directly look
at the gel on your illuminator: You´ll see orange bands on a black
background...

SURE: that sensitivity is neither "the ultimate sensitivity " nor "lower
than 0.1ng DNA", but in routine work you almost never have such low
amounts (Why should you, in the age of cloning and PCR?).

OK: there´s one application where destaining IS necessary: the
electrophoresis of RNA on formaldehyde-containing gels. 

Hope this helps
Frank



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