DNA extraction with phenol alone or phenol-chloroform - (Aug/24/2006 )
Hi all, i would like to know what is the difference by using phenol or phenol-chloroform for DNA extraction, sometimes i find protocols that use phenol alone and others that use phenol-chloroform...
I am going to paste in a quote from Phage434:
"Actually, I'd recommend that you at least wash with chloroform to remove residual phenol, which is highly inhibitory to downstream applications and also affects spectrophotometer readings. I would dilute this DNA 10-50x before I started to 100- 500 ng/ul, phenol chloroform extract once or twice, extract with chloroform, and then ethanol precipitate. Vortexing plasmid preps is usually not a problem.
also please note: vortexing small plasmids is usually no problem at all, but don't vortex large DNA or you are in danger of shearing. I would also say, I usually extract once with phenol, twice with phenol/chloroform/isoamyl alcohol 25:24:1, then twice with chloroform/isoamyl alcohol 24:1. You can 'do the experiment', though...try it several ways on the same overnight culture and run the results on a gel, and check them in your spec and see what you get?
A quote from http://www.protocol-online.org/biology-for...posts/7139.html.
"Phenol and chloroform are both organic solvents, so lysed cell components that're hydrophobic will be trapped in these solvents, e.g. membrane lipids, hydrophobic polypeptide sequences (protein) or polysaccharides etc. etc.
Both solvents are also powerful denaturants, therefore proteins will be denatured, leaving hydrophobic segments to interact with organic solvent and hydrophilic segment to interact with aqueous. This is why we see interphase (containing proteins or polysaccharides etc.) during organic extraction.
At the end, it leaves only hydrophilic entities in the aqueous solution, e.g. nucleic acids, sugar, salt, etc. etc..."
Now the answer...
Using phenol is advantageous because it separate proteins well but the drawback is that it's a little soluble in water (aqueous), so at the end, it'll contaminate your DNA. AS for chloroform, it's immiscible to water, so by combining chloroform with phenol, both will separate proteins well and at the same time, chloroform will ensure phenol stay organic and not wandered of to aqueous phase. Sometimes, we just do chloroform-isoamyl alcohol separation because we really want to ensure there's no residual phenol that is still in aqueous phase.