HELP! -- Protecting DNA on a transilluminator?
Frank O. Fackelmayer
fof1 at chclu.chemie.uni-konstanz.de
Tue Jul 29 10:42:35 EST 1997
In article <33dd3e14.1135487 at news.io.com>, lader at ambion.com (Eric Lader) wrote:
> On Mon, 07 Jul 1997 19:40:14 +0000, drm21 at mole.bio.cam.ac.uk (David
> Micklem) wrote:
> >In article <33c12035.5223697 at news.uchsc.edu>, Vern Shellman wrote:
> >>I am having problems ligating my DNA fragments cut out of a gel. I
> >>get variable results that seem to relate to how long it takes to cut
> >>out the band. The fastest I can see and cut out a band is about 15
> >>seconds and by then I suspect there is already significant UV damage.
> >>Is there any way to protect my DNA during UV exposure?
> >Use a LONG-WAVE UV transilluminator! If you haven't got a big long-wave
> >box, then use one of the little hand-help ones. (Of course, you might be
> >using long-wave already, in which case...):
> >I suppose you might gain a little time by attenuating the UV with
> >something, if you can't get hold of a long-wave source (but it'll be harder
> >to see the bands).
> >If you are purifying a LOT of DNA, and run the gel with a little ethidium
> >bromide in the gel and buffer, then you can often see the bands in daylight
> >against a white background. (This may also work with post-staining the gel,
> >but I haven't tried).
> >Alternatively, I think that the FAQ has methods for staining DNA without
> >using Ethidium bromide and UV - although it is less sensitive. The method
> >uses methylene blue IIRC.
> I read something about putting guanosine in the gel and running buffer
> making a profound difference. Don't remember the reference
You can speed up band excission to less then 2sec if you do it the
-Run your gel as normal, then stain with EtBr
-place your gel on the UV transilluminator, DO NOT TURN ON YET
-cut your gel in slices, between the lanes, so that you get slices
containing one lane of your original gel each.
-let the marker lane in place, and push the other lane slices away to the
area surrounding the UV window of your illuminator (so that they wont see
-then place the first lane slice side by side with your marker lane
-with a scalpel at hand, TURN ON the illuminator, locate your band, and
make two cuts above and below your band, then IMMEDIATELY turn off your
-put your cut-out band in a cup, then proceed with the next lane slice
-when you´re finished with all fragments, you can arrange the slices to
make a photo if you wish.
With a little practice, you get your desired bands with UV exposures less
then 2sec. I have been doing that for years, and never had any problems
with the isolated fragments. There is only one prerequisite for this
method: You must exactly know what you expect. Sometimes it is better to
use a small aliquot and run a minigel in advance, to see the fragments you
Hope this helps
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