staining DNA in agarose gels without EtBr

Paul N Hengen pnh at
Wed May 19 13:36:51 EST 1993

In article <1993May19.141214.8015 at>
 ahill at (Mr. A.F. Hill) writes:

>does anyone have a protocol for staining DNA in agarose gels using dyes rather than 
>using ethidium bromide?
>i have tried the protocol from TIG's published a
>while ago but didnt get very good results. i had
>destained the gel for over 6 hours and was still
>left with a lot of background.
>has anyone else had similar experiences?

Andy: As I found by looking into past articles by gopher, you actually
asked this question sometime back. While the majority of people who answered
your question suggested methylene blue, others suggested some kind of shadowing
method. Have you tried any of the other methods successfully?

The best answer to your question above was posted before, suggesting that
you should destain much longer than 6 hours when using methylene blue.
Perhaps even using a lower concentration of stain might help.

These clips from gopher...

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> From: srckbjg at
> Subject: Re: Methylene blue to stain agarose gels
> Date: 15 Apr 93 02:21:42 GMT
> >As part of a followup to these two methods of subcloning agarose
> >gel purified DNA fragments I have found that using methylene blue
> >to visualize the bands vs. EB UV light results in a 10-fold higher
> >efficiency in insert-containing clone isolation.  Simply stain
> >your agarose gel in 0.25% methylene blue in 0.1X TAE for 30 min
> >and destain in 0.1X TAE until you can see the band you want.
> Recent edition of TIG (9(2):pg 40) reported comparison of basic dyes
> incl. methylene blue for nontoxic staining of agarose gels.
> They found the best was to immerse the gel in 0.04% brilliant cresol
> blue in 20% ethanol for 15-60 mins, wash dH20 10s, 70% ethanol 5s, 
> twice more dH20 to detect 25ng of DNA.
> Subject: DNA staining in high schools
> Date: 14 Aug 91 16:10:19 GMT
> Tin Wee TAN (BCHTANTW at NUSVM.BITNET) called my attention to
> Berger and Kimmel, Meths Enzymol, Vol 152. ("Guide to Molecular Cloning 
> Techniques"), Academic Press, 1987, page 71, where Ogden & Adams describe 
> the use of methylene blue dye.  Their procedure:
> 1.  Stain in 0.02% methylene blue, 10mM Tris Acetate pH 8.3 for 1-2 hr at
>     4 deg C.
> 2.  Wash off excess stain with several changes of water over 5-8 hr.
> Ogden & Adams give a limit of detectability of about 250ng/1 cm band.
> I tested essentially this procedure on minigels (20 ml 1% agarose in a 2"x3"
> slab), with wells 4mm long.  I stained for 90-100 minutes in 100 ml 0.02%
> methylene blue, (20 mg in 100 ml of either 1/10 electrophoresis buffer or
> water); I stained at room temperature.  I then destained in 1 liter water.
> Bands were visible within a fairly short time, but were best after overnight.
> Bands with only 10 ng DNA were visible; on one gel I could see a band with only
> 3.5 ng!  The color was stable for many days in the weak dye solution resulting 
> from the destaining, even at room temperature.
> A couple responders mentioned Stains-All. I used it a long time ago, but it
> was less convenient because it had to be dissolved in an organic solvent.  My
> recollection is that it also faded with time, perhaps photobleaching.
> Two people mentioned using biotinylated DNA, with appropriate color development.
> This would be too costly and complicated for my purposes, but might be worth-
> while in a more advanced class.  The experience it would give students with
> enzymes was mentioned as a plus.
> Benedik (see below) mentioned silver staining.
> From: msw1633 at (WHITSITT, MARK STEVEN)
> Subject: Re: DNA staining without Ethidium
> Date: 20 Sep 91 03:25:44 GMT
> >       I was just browsing through a catalog I received from Midwest
> >Scientific where they advertise a product called "Nuclistain" which is a 
> >positive stain for the detection of DNA and RNA (double or single strand)
> >in agarose or polyacrylamide gels. Detection limit about 50ng. Visible
> >in normal light. 
> >       The vendor was  Midwest Scientific, Tel: 1-800-227-9997
> >       (except in St.Louis: 225-9997)
> One suitable method for the visualization of nucleic acids is to use methylene 
> blue.  Several protocols exist and can be found in the molecular cloning 
> handbook by Sambrook et al. as well as a variety of other places.  Basically, 
> the gel is soaked in 0.2% methylene blue/ 10mM Tris-acetate (pH 8.3) for 1-2 
> hours at 4 degrees C avoiding direct sunlight (ie. the container is covered).
> Remove the excess stain by soaking in several changes of distilled water until 
> the bands become visible, again avoiding sunlight.  The limit of detection is
> around 250ng/1cm band.  This protocol comes from:  Guide to Molecular Staining 
> Techniques. Berger SL, and Kimmel AR eds. (Methods of Enzymology vol 152.
>  pp 71.)
> From: wjb1 at (William J. Buikema)
> Subject: Re: Suggestions for visualizing DNA
> Date: 13 Aug 91 17:36:58 GMT
> A postdoc in the lab here has used something called "Stains-All" from Kodak 
> for visualizing DNA oligos.  Information about the stain can be found in the
> Electrophoresis section of the Kodak catalog.  It will stain DNA, RNA, proteins
> and acidic polysaccharides, all different colors, and has somewhat less 
> sensitivity than ethidium (1/5th?).  The downside is that the staining 
> solution is made up in formamide and water.  

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

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