Size of genomic DNA

[Smriti]

The genomic DNA that we buy from manufacturers like Sigma, Roche etc. are always described in range of 100-50kb. However, we know that the actual genomic DNA is always much larger than this. Can anyone explain this? Is it something related to conformation of DNA migrating in gel?

[bbeitzel]

Large DNAs fragment during standard purifications. In order to obtain very large (Mb size) fragments, the DNA has to be handled very gently. I'm not an expert, but the one time I tried to run a pulsed-field gel we had to embed cells in agarose and then SDS/proteinase K treat the embeded cells. The agarose stabilizes the long DNA strands and allows you to see them on a gel at their full sizes. Don't know if that is still the standard procedure.

[Smriti]

Thanks for the quick reply! However, I wanted clarification on large DNA (>Mb size) migrating at 20-100kb band in 1% agarose gel. How can we relate both the sizes?

[HESmith]

Two reasons:

1) the DNA is not intact but broken into fragments.
2) standard gel electrophoresis cannot resolve Mb-sized DNA.

[Smriti]

Thanks HESmith for the clarification.

Wanted to know another fact regarding treatment of DNA at higher temperatures. We'll know that DNA denatures above 90 degrees. Lately, someone told me rising temp. above 50 degrees cause DNA to become AT-rich/bias. However, I could not find any literature evidence for this statement. Is that true?

[HESmith]

It's true. See the Quail et al., 2008, Nature Methods article.

[Smriti]

Thanks HESmith for recommending the article. Indeed, it was very helpful.