phenol-chloroform-extraction - (Sep/26/2005 )
Dear all,
as we use phenol-chloroform-isoamyl during DNA or RNA extraction and plasmid midi preparation, what is the actual mechanism to get rid of the protein by the phenol? is it related to the phenol molecule structure?
Thanks .......
hi
from ambion's website :
Efficient extraction of cell extracts or solutions containing nucleic acid are most often performed with a series of phenol and phenol:chloroform extractions at a specific pH. Both phenol and chloroform cause proteins to become denatured and become soluble in the organic phase or interphase, while nucleic acids remain in the aqueous phase. After centrifugation, the aqueous phase containing nucleic acid is re-extracted with an equal volume of chloroform:isoamyl alcohol (1,3,4). This combination of extractions is thought to reduce the loss of RNA due to the formation of insoluble protein:RNA complexes at the interphase.
Chloroform is mixed with phenol to increase the efficiency of nucleic acid extractions by reducing losses of RNA at the interphase. The increased efficiency is due to chloroform's ability to denature proteins and aid in the removal of lipids, thus improving separation of nucleic acid into the aqueous phase. Phase separation is also enhanced, which assists in the removal of the aqueous phase with minimal cross contamination from the organic phase. Often isoamyl alcohol is added to phenol:chloroform to reduce foaming
fred
Are we saying tha the RNA is isolated at the interphase and not lost in the aqueous phase, thus making a purer DNA or are we saying that the RNA is not lost to the interface (that is we are purifying RNA and not DNA)?
Thanks
hi
Ambion's is a great site for RNA stuff
Well answer to your question depends on the pH of the phenol solution. A pH 8 will drive the DNA extraction and RNA will be majoritary loss and you'll get DNA in aequous phase.
If pH is 4.7, we are talking of RNA.
Then the DNA is not able to get the aequous phase and you'll get RNA.
Ambion's is a great site for RNA stuff
Well answer to your question depends on the pH of the phenol solution. A pH 8 will drive the DNA extraction and RNA will be majoritary loss and you'll get DNA in aequous phase.
If pH is 4.7, we are talking of RNA.
Then the DNA is not able to get the aequous phase and you'll get RNA.
what is differant if we use (Phenol:Cloroform:octanol) and (Cloroform:octanol) on Phenol extraction?
phenol solution pH 8?
well i assume alcool is a kind of stabilizant... so IAA or octanol...if it works, go.
phenol pH 8 : it's phenol solution saturated with Tris pH 8
phenol is an organic solvent... hydrophobic!. so disrupts the 3 D structure of proteins. precipitates them.. phenol s used to create a good interphase as phenol is slightly soluble in water..
i think they are used in the ratio 25:24:1(phenol:water:chloroform).
and about the ph thingy. and selective extraction of DNA. my friends have got it right
i think they are used in the ratio 25:24:1(phenol:water:chloroform).
and about the ph thingy. and selective extraction of DNA. my friends have got it right
I think you meant phenol: chloroform: isoamyl alcohol at 25:24:1. What I read about isoamyl alcohol's function at Molecular Clonging is that it inhibits RNase, and usually an additional chloroform is used at the end to remove residual phenol from aqueous phase, which is slightly soluble and a known PCR inhibitor.
What I learn about pH is also similar. at pH 8, DNA is soluble, so it stays at aqueous phase, while at pH 6 or lower, DNA is less soluble and thus will go to organic phase and only RNA stays at aqueous phase. Based on this, I think RNA can still stay at aqueous phase at pH8. These are what I've heard, not from any text.
phenol is an organic solvent... hydrophobic!. so disrupts the 3 D structure of proteins. precipitates them.. phenol s used to create a good interphase as phenol is slightly soluble in water..
i think they are used in the ratio 25:24:1(phenol:water:chloroform).
and about the ph thingy. and selective extraction of DNA. my friends have got it right
I think you meant phenol: chloroform: isoamyl alcohol at 25:24:1. What I read about isoamyl alcohol's function at Molecular Clonging is that it inhibits RNase, and usually an additional chloroform is used at the end to remove residual phenol from aqueous phase, which is slightly soluble and a known PCR inhibitor.
What I learn about pH is also similar. at pH 8, DNA is soluble, so it stays at aqueous phase, while at pH 6 or lower, DNA is less soluble and thus will go to organic phase and only RNA stays at aqueous phase. Based on this, I think RNA can still stay at aqueous phase at pH8. These are what I've heard, not from any text.
I have been using just chloroform while extracting genomic DNA from yeast S. cerevisae. Is this the right way to go about it? Should I add phenol to it as previous posts say? Is chloroform alone enough to have the proteins denatured in between the two phases?
Best Regards
Chloroform is very effective at removing lipids, but will do little to remove protein contamination.