Boiling ethidium bromide gel
svetlov at oncology.wisc.edu
Mon Apr 27 13:09:26 EST 1998
In article <REPLACE-2504981632560001 at macbb2212b.unil.ch>,
REPLACE at lbm.unil.ch (Stephan) wrote:
> In article <svetlov-ya02408000R2404981218530001 at news.doit.wisc.edu>,
> svetlov at oncology.wisc.edu (Vladimir Svetlov) wrote:
> > Recently I've taken up
> > adding EtBr to the loading buffer instead of the gel - far less EtBr to
> > worry about (in the running buffer etc.), lower background and you can boil
> > the agarose as much as you want.
> Mmmm... I'm a little bit sceptical about that method. Can you really see
> small fragments? (let's say, smaller than 1 kb?). What is the minimal
> amount of DNA that you can detect? (let's say, can you see well all the
> bands when you load a 0.1 ug sample of lambda/HindIII?).
> If this method really works, I would be very interested to know more about
> it (how much EtBr to your sample?).
BIO101 actually sells such loading dye under the name of the Green Dye -
your normal ficoll-based stuff with EtBr in it. Inserts down to 200 bp
appear quite bright and on the plus side you don't have to run EtBr out of
the gel to reduce the fluorescent background, obscuring the small, low
intensity bands. When we ran out of the "green dye" I've started making me
own by adding 1/10 of the volume of EtBr to the sucrose-based BrPh/XyCya
dye premix. The bottle of EtBr I get it from dates back to the Reagan era
so I don't really know how close the 100 ug/ml is to the reality, but it
works OK as it is. I then added some EtBr (a pinch, as them French would
put it) into my 1 kb DNA ladder and I'm using it for couple of months
already without noticeable changes in bands appearance.
There are other methods to deal with the excess of agarose, such as cutting
up the gel into slices and keeping them at 4-8 C in a plastic bag with some
buffer. 1.5-2% gels can be stored like that for several weeks without
loosing the resolution capacity or EtBr. Gels with lower than 1% of agarose
can sometimes collapse the wells but normally they come right back at you
when you flood them with the running buffer. Some people store molten
agarose at 55 C but it's good only for a few days because a) stuff like
NuSieve does not take it very well and gel comes out very brittle and b)
them dumbasses forget about it and agarose with time turns yellow, brown
and eventually starts resembling coal. Then you have to boil it with NaOH
to get this shit off the glass.
Vladimir Svetlov, Ph. D.
McArdle Lab for Cancer Research
1400 University Ave.
Madison, WI 53706
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