Methylene Blue DNA staining protocol

Duncan Clark Duncan at genesys.demon.co.uk
Wed Dec 29 14:10:00 EST 1993


Hi Netters,

A number of people have requested the protocol for staining DNA with
methylene blue. This is a copy of the original post a few weeks ago. It works
very well.


As a followup to the methylene blue staining discussion
just begun (and from a few months back), I have found that
a slight modification greatly reduces the background
staining problem.  Instead of staining in 0.02% methylene
blue for 30-60 min and then destaining for what seems to
be forever I've found that 0.002% (1/10th X) works just as
well and the background is much lighter.  I have also found
much to my surprise that NuSieve:Agarose (3:1, 4% final) gels
stain very nicely and that dsDNA as small as 75 bp is easily
visualized.  I'm currently using MB staining for all of
my cloning work as I discovered that I get 20-50X as many
positive clones from band isolated DNA when comparing MB
to ethidium bromide/UV transillumination.
     Hope that this was of some help
                              Eric
--
  Eric R. Hugo, Postdoctoral Research Associate |eric at bcserv.wustl.edu
  Dept. of Biochemistry and Molecular Biophysics|finger above for            
  Washington University School of Medicine      |public key <-- 
  Box 8231, St. Louis, MO 63110_________________| (314) 362-3342


Dear Bionetters,
     My recent followup to a DNA staing question has 'netted'
me several email requests to post a more complete protocol
for MB staining DNA.  Here it is:


               Methylene Blue DNA stain
Protocol:

1.  Load 2-5X the amount of DNA that would give bands of moderate
    intensity on an ethidium bromide stained gel.  Typically this
    is something on the order of 0.5-2.5 ug of a 1 kb fragment on
    a 30 ml 1% mini gel.  These numbers are guess-timates so your
    milage may vary.

2.  Run the gel normally and then place in a 0.002% methylene blue
    (w/v, Sigma M-4159) solution in 0.1X TAE (0.004M tris 0.0001 M
    EDTA) for 1-4 h at room temp (22 C) or overnight at 4 C.  Diffusion
    of the DNA does not seem to be a problem for fragments as small
    as 100 bp (3% Nusieve:1%agarose gel).

3.  If destaining is needed to increase the visibility of the bands
    place the gel in 0.1X TAE with gentle agitation changing the buffer
    every 30 - 60 min until you are satisfied with the degree of
    destaining.

Notes:

     I performed a side by side set of experiments to compare ethidium
bromide/UV to MB staining.  All though I only did the experiment once
the results were surprising.  DNA isolated from MB stained gels
transformed frozen competant E. coli (XL1-Blue and DH5) on the order
of 20-50 fold more efficiently than EB isolated DNA.  These numbers
are not quite as precise as I'd like because I got so FEW colonies /plate
with the EB material.  I suspect the differences you will get will
depend on a variety of factors such as: how fast you cut out the bands,
output spectrum and intensity of your transilluminator, and the %AT of your
DNA to mention a few.  One of the advantages of MB staining is the
elimination of several of the variables.
     I have tried the staining procedure with several types of
agarose.  Both FMC GTG agarose and Nusieve GTG perform very well.
I tried another sieving gel product called Synergel and found it to
be incompatible with MB (very high background).  According to the
published info MB staining is very compatible with polyacrylamide
(even less of a background problem) but I haven't had any experience
in this matter.
     One individual asked if the method was quantitative.  I've
never tried to quantitate but I suppose the easiest way to do this
would be to excise the stained band, dissolve in 6 M NaI, and
read in a spectrophotometer.  One would have to be very careful
to excise the exact same amount of gel for each band (using maybe
a cork hole borer) and to run internal standards but I think it
would work at least in a semi quantitative fashion.
        I guess the other big advantage this method has over EB/UV
is the avoidance of UV skin burns.  I don't know about the rest of
you but I got a fairly severe burn as a naive young grad student.
After isolating 28 bands from I gel I realized I was in big trouble.
Within a couple of hours my face was beginning to peel off.  Fortunately
I had eye protection on but that certainly didn't protect very much
surface area.  That was a painful lesson to learn (1st time I'd ever
been burned INSIDE my nose!) and one that I was very quick to avoid.
As far as I'm concerned UV exposure is as big if not bigger a hazard
as the ethidium bromide itself.  Methylene blue is not nearly as toxic
although I just noticed that the bottle claims that it is a possible
mutagen (maybe they put this on anything that can interact with DNA ?).
     If I get a chance and have an interested undergrad wanting
to do a quick project, I'll try to get more quantitative data
comparing the two methods at my new job in N. Dakota.  If anyone
out there has anything to add to the methylene blue story (ie.
good or bad experiences) please send me email.  If I get enough
responses I'll post a summary.
     One other quick note,  Wayne Barnes, here at WUMS suggested
that one might be able to eliminate the difference in transformation
efficiency between EB and MB isolated DNA by inducing a SOS response
in the competant cells.  He suggests briefly irradiating the E. coli
with UV before making them competant.  I haven't tried this but if
anyone has I'd like to hear about your results.  Personally, making
HIGHLY competant E. coli requires enough lab voodoo as it is without
adding even more variables.
                              Eric
--
  Eric R. Hugo, Postdoctoral Research Associate |eric at bcserv.wustl.edu
  Dept. of Biochemistry and Molecular Biophysics|finger above for            
  Washington University School of Medicine      |public key <-- 
  Box 8231, St. Louis, MO 63110_________________| (314) 362-3342


-- 
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Duncan Clark                        | Internet:    duncan at genesys.demon.co.uk
GeneSys Ltd.                        | Compuserve:  100015.1406 at compuserve.com
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