problem about DNA electrophoresis - the DNAs become two layers in the gel (Jun/28/2010 )

Dear everyone, I have a problem about the DNA electrophoresis. when I use the big well to run the DNA electrophoresis(in order to extraction the pcr product from gel), the sample can not settle down completely. The DNAs become two layers and there are no DNAs in the middle of the gel. When I use the small well, everything is ok. I do not know what happened, could you give me some suggestions! Thanks very much!

i'm not sure if i got it right: is it like the edges of your band are stained and not the centre? Then maybe it's because you loaded a much higher amount of DNA in the big well and the dye you are using doesn't stain the centre of the band because there's so much DNA? Do you have a picture?

tantao on Jun 28 2010, 11:45 AM said:

Do you mean you see two bands with an unstained middle when looking at the gel from the top (i.e. with the gel in the orientation it's in when you load it) or when looking at the gel slice in cross-section?

If the former, then you have two different DNA molecules present in your sample which have been separated by size through electrophoresis. If the latter, then it's likely the stain didn't penetrate the gel completely, and this staining artifact is of no consequence, but likely could be overcome by staining the gel longer to allow complete penetration of the gel.

I noticed the same phenomenon. It is because of post-straining (with GelGreen or SybrGreen). The dye diffuses into the gel very slowly from both sides and does not reach central part of the gel. The bands are strained only at the top and the bottom (when viewed from profile). You can be sure that your DNA is there when you excise it from gel, you just do not see it.

Thanks very much, fysio lab ,home brew and vladooo. The phenomenon is that I see two bands with an unstained middle part when look at the gel slice in vertical cross-section. So I think I should strain the gel more longer. Thanks everyone again !