In article <aldridge-ya023480002301971031250001 at news.unimelb.edu.au>,
aldridge at mbcmr.unimelb.edu.au (David Aldridge) wrote:
> On 12 Jan 1997 13:40:33 GMT, "Thorsten Schmidt"
> <Thorsten.Schmidt at rz.ruhr-uni-bochum.de> wrote:
>
> >Is there anything to be said against preparing a 3 % and a 10 % gel-mix
> >by mixing the componets (except for APS & TEMED) in advance?
> >
> >It would minimize time by just adding TEMED & APS to a aliquot!
> >Or isn´t it good to store such a mix for a longer time?
>> Acrylamide is unstable and will break down, by deamination, to acrylic acid
> in a reaction catalysed by light and alkali. I'm not sure of the details,
> but by checking the pH of and old acrylamide solution will give an
> indication of whether it has broken down yet. Also adding a small amout of
> basic ion exchange resin will help preserve it. You should therefore not
> add the acylamide to your mix until ready to use it. The Tris, water and
> SDS can however be mixed in advance.
Here are some empyrical experiences. In our lab we have done this. For
the separating gel I would not recommend this, although a premixed
solution can be used for several days, the gel properties change and the
separation gets worse quickly. Anyway it really doesn't save much time.
you can make up a 5x SDS buffer and it takes minutes to mix acrylamide
buffer and water. The stacking gel solution is not as sensitive. I have
used a couple of months old premixes (stored in the fridge) and the gels
look just fine, the down side is that old mixes take longer to polymerize
and thus the minutes you save by premixing are eaten up and more than
that! This makes sense if the reaction is catalyzed by alkali, since the
pH of the stacking gel buffer is lower and one would therefore expect the
deamination to be slower than in the separating gel mix.
The funny thing is that I routinely make 6% premixes (with Urea) for
sequencing gels and have never had any problems storing them. I have made
beautiful gels with several months old solutions stored in a transparent
glass bottle at 4*C. Why does the acrylamide hydrolyze so much faster in
the SDS buffer? The pH difference is not that big, both are above pH 8.
Any ideas?
Zophonias
_____________________________________________________________________
Zophonias O. Jonsson
Institut fur Veterinarbiochemie Tel: (41-1)-257-54-75
Universitat Zurich-Irchel Fax: (41-1)-362-05-01
Winterthurerstrasse 190
CH-8057 Zurich
Switzerland
_____________________________________________________________________
