A couple of people in our lab add ethidium bromide solution to hot agarose
(i.e. nearly boiling), and claim that ethidium bromide cannot escape as a
gas so there is no danger. This runs contrary to everything else I have
heard, which says the agarose solution should be left to cool first, or
the etbr added in a fume cupboard. Obviously this later method seems to be the
safest, but is there any merit to their claim?
"[Only registered users see links. ].uk" <[Only registered users see links. ].ac.uk> writes:
Ethidium bromide has completely negligible vapor pressure at 100C. I
*suppose* you might be concerned about it splashing. But why are you
so concerned about below-detectable-limit EtBr when there are *much*
more hazardous things in your laboratory, such as chloroform,
formaldehyde, or phenol? My theory is that people worry because they
can see high concentrations of it.
If you add it to hot agarose, you will degrade it (lower sensitivity).
But of course you are adding a lot anyway, so it won't really affect
your results too much...it boils down to a time and convenience issue.
Speaking of which...I'm very impatient. So I usually cool hot agarose in
a cold water bath with constant stirring (usually about a minute). And
then when it has cooled enough to place on your cheek without "hurting",
then I pour. If I'm really pressed, and if I can find the space, I put
the gel into a fridge or freezer until it has solidified.
You add it in a fumehood or a designated area to limit exposure.
Sometimes, powder can line the bottle cap of a solution too, so you
might cause contamination this way.
The other thing to keep in mind is that any intercalators like EtBr will
affect the eletrophoretic properties of DNA when it is included in the
running buffer or the gel. So it is important to think about your
application to determine how best to stain.