Doesnt the histone get seperated during DNA extraction? - DNA extraction (Jul/27/2005 )
While DNA extraction, when phenol:chloroform is used or when proteases are used to get rid of the proteins in the cell extract, what happens to the histones associated with the DNA? Doesnt they get removed too?
Also I read at some website that mRNA get seperated in the middle seperation layer while phenol:chlroform extraction of the DNA, if phenol extraction removes just the lipids and proteins, how does mRNA get seperated?
Yes, I believe that histones are removed by proteinase K digestion and/or phenol chloroform extraction.
As to the mRNA seperation thing, perhaps they were talking about acidic phenol or something? You can use phenol pH~5.2 to extract RNA I think this makes the DNA go to the interface to give cleaner RNA preps.
I never heard of it before, but when using neutral pH phenol (7-8) to isolate DNA maybe the opposite happens??? Anyone know??
I accidentally used TE-saturated phenol, pH 6.6 in a DNA extraction last week and hardly had any template left for the run-off transcription reaction that I wanted to do. Using phenol < pH 7.0 in DNA extractions will result in very low yields as the DNA will partition into the interface or organic phase.
Check out Ambion for a references:
RNA is in the organic phase. If you want to extract RNA a pH 4.7 phenol should be used.
Hank is right: pH is case sensitive in these extractions of nucleic acids and good extraction of proteins from DNA...
If histones get removed it should affect the DNA conformation.....since DNA is bound on the histones. What could be the conformational changes?wouldnt it make it more susceptible to be sheared ?? I am trying to think at the molecular level the changes that can be expected to occur in the DNA conformation. Does anyone know? or can refer me to a website that will give me some info??
i think DNA fold itself by some affinity bounds, but it's quite un previsible.
Regarding genomic DNA, a denaturation step before exp destroy these foldings, and for DNA minipreps, at 37°, these interactions are too weak for resist. I think the weakness of these foldings allows experiences not to be jam.