Why don't we stain the DNA before separation by Gel Electrophoresis?

[Kara]

After reading the lab III-7a download, I am wondering why we couldn't dye the DNA with methylene blue before running the gel electrophoresis.  Also, will gel electrophoresis work using complete DNA molecules?

I ask because I was considering running DNA from several sources side-by-side without breaking the DNA into smaller chunks.  Presumably this would provide a single well-defined band for each DNA type with different DNA types making different rates of progress across the gel depending on the overall size of the DNA molecules. Wouldn't pre-dying would allow us to track the progress without the need for a marker dye?  Are full-size DNA molecules simply too large to make it through the gel?

Thank you, Kara

[The Home Scientist]

Hi, Kara

Good questions, and I don't know the answers for sure because I've never done it that way. Why not give it a try and report back what you observe?

As to staining, my guess is that the methylene blue won't be bound tightly enough to the DNA to migrate with it through the substrate. The purpose of the marker dye isn't to bind to the DNA but rather to move independently through the agar substrate at about the same rate as the small fragments you're trying to separate. When the marker dye nears the far end of the substrate, the smaller DNA fragments should be near it, with the larger fragments strung out behind and the largest still very near the original wells.

Staining after you run the separation identifies the bands by staining them in place, so you don't need to worry about the dye migrating separately. But again, don't take my word for it. Run it both ways and see for yourself.

As to running whole DNA, my guess is that you'll run into difficulty trying to get it to move through the substrate at all, but that depends a lot on the type (size) of the DNA molecules, the voltage you're using, and many other factors.

Best regards.

Bob 

[Kara]

Bob,

Thanks for your thoughtful reply.  We are still several months out from running the gel electrophoresis lab with our daughter but now hope to have enough time and resources to follow your advice and run it both ways.  If so we'll certainly report back with our results.  

Thanks again,

Kara