6 replies to this topic

[Tfal]

The Bio-Rad Mini-Protean 3 Manual indicate to add Running (separating) gel buffer on to of gel if it is to be used next day. The way it is is indicated, it seems you do not cast the pre gel (stacking) until next day, before running electrophoresis.
I used to cast both separating and stacking gels, leave the comb in keep it in hermetical bags (without buffer or water) up to a couple of weeks? Is this ok?
Should I stick with Bio-Rad's guidelines?

[medchemgirl]

I would call Bio-Rad tech support, they will know for sure.

Tfal, on May 20 2009, 11:20 AM, said:

The Bio-Rad Mini-Protean 3 Manual indicate to add Running (separating) gel buffer on to of gel if it is to be used next day. The way it is is indicated, it seems you do not cast the pre gel (stacking) until next day, before running electrophoresis.
I used to cast both separating and stacking gels, leave the comb in keep it in hermetical bags (without buffer or water) up to a couple of weeks? Is this ok?
Should I stick with Bio-Rad's guidelines?

[T C]

Hey,

I cast the gels before hand, put them in a cling film with water and store at 4 degrees for 1-2 weeks without any problem.


best,
TC

Tfal, on May 20 2009, 10:50 PM, said:

The Bio-Rad Mini-Protean 3 Manual indicate to add Running (separating) gel buffer on to of gel if it is to be used next day. The way it is is indicated, it seems you do not cast the pre gel (stacking) until next day, before running electrophoresis.
I used to cast both separating and stacking gels, leave the comb in keep it in hermetical bags (without buffer or water) up to a couple of weeks? Is this ok?
Should I stick with Bio-Rad's guidelines?

[little mouse]

I can cast the separative gel several days before, but I prefere to cast the stacking gels the day of the migration. They have two different pH. I don't know if it's right, but I have the feeling that when I poured the stacking gel in advance, the high molecular bands were less focused.

[Irene G]

Hi,
we have done both, preparing all the gel the day of the migration and do it the day before, or two or three days before, but when we prepare them previously we keep the gel at 4ºC and we envelop it with paper soaked in the migration buffer. We have checked that the second way (one or more days before, though we haven't try more than 3 days) give more defined bands.
Hope this can help you.
Irene

[Penguin]

The problem with making gels in advance and storing them is that you can lose the pH gradient between the stacking and resolving gels i.e. the pH 6.8 Tris will diffuse out and mix with the pH 8.8 Tris.  The stacking gel is at a lower pH than the resolving gel to increase resolution (see link) which is why you may be seeing fuzzy bands if you don't use freshly made gels.

http://www.promega.c...0507/fq0043.htm

P

[Tfal]

thanks for all the advice

so it seems to make sense to precast the separating gel and add the stacking gel the day of the migration, to make sure to preserve the pH gradient between the two gels.

about the buffer in which one keeps the gels, Bio-Rad indicates 1.5 M Tris-HCl pH 8.8, which makes sense as long as you only have your separating gel. Anyway, that's what I've been doing lately and it seems ok.
I guess that if one really wishes to precast both gels (sep. & stack.), electrophoresis (migrating) might be sensible choice.  1.5 M Tris-HCl pH 8.8 might interfere with the stacking gel.

thanks again,
Tfal.