ethidium bromide: add to gel before pouring or stain afterwards
Frank O. Fackelmayer
fof1 at chclu.chemie.uni-konstanz.de
Mon Jun 29 03:04:03 EST 1998
Aniko Varga wrote:
> thx for the ideas...
> we use our gels a max of three times to cut costs with the high res
> gels... run the dna and etbr off before melting, touched-up with some
> distilled water, not buffer (to avoid too salty of gels) to bring back
> to initial volume
> destaining is a hassle... especially with large gels, moving them from
> bath to bath... any easy ways to do this, especially with large gels?
We are always staining our gels after the run. I cannot understand what the
problem is with that. There is definitely no need for destaining, it does not
take much time, and it is safe. I guess that people have to destain their gels
only because they use WAY to much EtBr. As a rule of thumb: if your staining
solution appears slightly orange, you use to much. A good staining solution is
colorless, or VERY VERY slightly orange when viewed against white background.
I add 10ul of EtBr (stock 1%, i.e. 10mg/ml) to 250-500ml of running buffer
(TAE or TBE), and stain the gel in this solution for 15min, then rinse briefly
(20sec) with tap water. I easily see bands with 5-10ng of DNA, so there is no
problem with sensitivity. Staining solution can be re-used at least 10 times
before a marked drop in sensitivity occurs.
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