gel electrophoresis

[sammy123]

why does the bands in agarose gel turn rough and fuzzy if left wrapped with a plastic in tbe buffer in refrigerator after some days? how are the bands sharp and defined at first?!


New science geek

[danfive]

The DNA molecules, or pcr amplicons hold the intercalating agent, EtBr, after electrophoreses they are focused, but as time goes by the slowly diffuse, you can actually refocus by putting back into electrophoresis apparatus for a few minutes (but that would contaminate the electrophoresis box with EtBr, a carcinogen).

[sammy123]

Thank you
Could be a bit detailed please?! I did not understand what you mean by them being focused???1

[danfive]

Originally Posted by sammy123

Thank you
Could be a bit detailed please?! I did not understand what you mean by them being focused???1

Electrophoresis--applying electricity to charged molecules causing them to migrate at different pace through the gel medium, and thus separating from each other based on size and total charge. The identical molecules are thus sharply grouped, aligned i.e focused (based on shape/charge) after the electrophoresis run.

If you want them to become sharp and compact, apply more electrophoresis.

[pigfarmer]

Originally Posted by danfive

The DNA molecules, or pcr amplicons hold the intercalating agent, EtBr, after electrophoreses they are focused, but as time goes by the slowly diffuse, you can actually refocus by putting back into electrophoresis apparatus for a few minutes (but that would contaminate the electrophoresis box with EtBr, a carcinogen).

Have you ever done this (refocusing)? It don't believe that would work, as I re-use gels quite often, and the bands do not refocus.

[danfive]

Originally Posted by pigfarmer

Have you ever done this (refocusing)? It don't believe that would work, as I re-use gels quite often, and the bands do not refocus.

I've done it within a few hours of the original run. I also always loaded only 10ul of 25ul rxn, so I could and would re-do the entire run if necessary.