RNA extraction-PCI method-how to shear genomic DNA - method (May/19/2008 )
Hi guys, I have a general question about RNA extraction.
I normally use acidic phenol-chloroform-isoamyl to extract RNA samples from E. coli cells. I understand that genomic DNA at Ph 4.3 should be extracted into the phenol phase while RNA remains in the aqueous phase. However, sometimes I got really pure RNA without any DNA contamination but other times I had sheared DNA in my RNA samples. So I think, when genomic DNA is intact, the size and the viscosity of DNA should make it easily be separated from RNA. However, when DNA is sheared into small pieces, it would be much harder for DNA-RNA separation, even at acidic Ph.
I am just wondering, does anyone know how to shear bacteria genomic DNA on purpose? Like, vortexing at XXX speeds or inverting tubes XXX times?
There must be something subtle in my procedure that causes sheared DNA....a mystery
Pipet tips with small holes and fast expulsion will shear DNA very effectively. Use wide bore tips and pipet slowly if you want to avoid shearing DNA.
As phage mentioned, narrow gauge pipette tip would be a big culprit. You shouldn't do vigorous vortexing ( I don't do any), and vigorous shaking of tubes. Many dead cells (especially in eukaryotic cell culture) would also give a lot of degraded genomic DNA.
I would anyways suggest DNase treatment for inter-sample uniformity; especially if you are going for a sensitive, quantitative, comparative assays like Real-time and microarray.
Edit: By the way, I have seen that stressed guys end up with sheared DNA more often.